Hafnia alvei based pharmaceutical and food compositions for inducing satiation and prolonging satiety

ABSTRACT

The present invention relates to pharmaceutical and food compositions for inducing satiation and prolonging satiety in subjects in need thereof. In particular, the present invention relates to a method of inducing satiation in a subject in need thereof comprising administering to the subject an effective amount of Hafnia alvei.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/EP2017/058116, filed Apr. 5, 2017,which designates the U.S. and which claims priority to InternationalApplication No. PCT/IB2016/051923, filed Apr. 5, 2016, the contents ofeach of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 5, 2018, isnamed 20181005_692-PCT-3-Sequence_Listing_080439-093610USPX.txt and is7,782 bytes in size.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical and food compositionsfor inducing satiation and prolonging satiety in subjects in needthereof.

BACKGROUND OF THE INVENTION

The composition of gut microbiota has been associated with hostmetabolic phenotypes (Ley et al., 2006) and transfer of “obese”microbiota can induce adiposity (Turnbaugh et al., 2006) and hyperphagia(Vijay-Kumar et al., 2010), suggesting that gut microbiota may influencehost feeding behavior. Although the mechanisms underlying effects of gutbacteria on host appetite are unknown, it is likely that they may usehost molecular pathways controlling food intake.

The current model of food intake control implicates gut-derived hungerand satiety hormones signaling to several brain circuitries regulatinghomeostatic and hedonic aspects of feeding (Berthoud, 2011; Inui, 1999;Murphy and Bloom, 2006). Prominent amongst these are the anorexigenicand orexigenic pathways originating from the hypothalamic arcuatenucleus (ARC) that include the proopiomelanocortin (POMC) andneuropeptide Y (NPY)/agouti-related protein (AgRP) neurons,respectively, relayed in the paraventricular nucleus (PVN) (Atasoy etal., 2012; Cowley et al., 1999; Garfield et al., 2015; Shi et al.,2013). The ARC and PVN pathways converge in the lateral parabrachialnucleus which sends anorexigenic projections to the central amygdala(CeA), expressing calcitonin gene-related peptide (CGRP) (Carter et al.,2013; Garfield et al., 2015).

Putative mechanisms of gut microbiota effect on host control of appetitemay involve its energy harvesting activities (Turnbaugh et al., 2006)and production of neuroactive transmitters and metabolites (Dinan etal., 2015; Forsythe and Kunze, 2013; Sharon et al., 2014). The inventorsevidenced an implication of bacterial proteins which act directly onappetite-controlling pathways locally in the gut or systemically. Infact, several bacterial proteins have been shown to display sequencehomology with peptide hormones regulating appetite (Fetissov et al.,2008), and recently ClpB protein produced by gut commensal Escherichiacoli (E. coli) was identified as an antigen-mimetic ofα-melanocyte-stimulating hormone (α-MSH) (Tennoune et al., 2014). a-MSHis a POMC-derived neuropeptide playing a key role in signaling satiationby activation of the melanocortin receptors 4 (MC4R) (Cone, 2005).Although MC4R-mediated α-MSH anorexigenic effects have been mainlyascribed to its central sites of actions (Mul et al., 2013), a recentstudy showed that activation of the MC4R in the gut enteroendocrinecells stimulates release of satiety hormones glucagon-like peptide-1(GLP-1) and peptide YY (PYY) (Panaro et al., 2014). Thus, gutbacteria-derived α-MSH-like molecules can directly act onenteroendocrine cells synthetizing satiety hormones.

SUMMARY

The present invention relates to pharmaceutical and food compositionsfor inducing satiation and prolonging satiety in subjects in needthereof. In particular, the present invention is defined by the claims.

The present invention relates to a method of inducing satiation in asubject in need thereof comprising administering to the subject aneffective amount of Hafnia alvei.

The present invention further relates to a method of prolonging satietyin a subject in need thereof comprising administering to the subject aneffective amount of Hafnia alvei.

The present invention also relates to a method of reducing meal size ina subject in need thereof comprising administering to the subject aneffective amount of Hafnia alvei.

Another object of the present invention is a method of reducing foodintake in a subject in need thereof comprising administering to thesubject an effective amount of Hafnia alvei.

The present invention further relates to a method of controlling weightgain in a subject in need thereof comprising administering to thesubject an effective amount of Hafnia alvei.

A further object of the present invention is a method of stimulatingweight loss in a subject in need thereof comprising administering to thesubject an effective amount of Hafnia alvei.

In one embodiment, the subject is not obese. In another embodiment, thesubject is obese.

In one embodiment, Hafnia alvei is administered to the subject in theform of a pharmaceutical composition. In another embodiment, Hafniaalvei is administered to the subject in the form of a food composition.

In one embodiment, the food composition is selected from the groupcomprising complete food compositions, food supplements, andnutraceutical compositions.

In one embodiment, Hafnia alvei is a probiotic. In another embodiment,Hafnia alvei is inactivated.

In one embodiment, the food composition comprises at least oneprebiotic. In one embodiment, the at least one prebiotic is dietaryfibers.

DETAILED DESCRIPTION

The inventors have demonstrated that the ClpB protein or a bacteriumthat expresses the ClpB protein is capable of inducing satiation,prolonging satiety, reducing food intake, controlling weight gain,stimulating weight loss and/or reducing fat mass on lean mass ratio in asubject in need thereof. The inventors have indeed surprisingly shownthat the ClpB protein expressed by said bacterium had a direct action onsatiation, satiety and food intake, probably via MCR receptors,independently of any immune reaction that could be induced by bacteriaadministration.

Accordingly, one aspect of the present invention relates to a method ofinducing satiation in a subject in need thereof comprising administeringto the subject an effective amount of a ClpB protein or variant thereof,or an effective amount of a bacterium that expresses the ClpB protein orvariant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for inducing satiation in a subject in needthereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forinducing satiation in a subject, in particular in a subject havingnormal weight or uncomplicated overweight.

One further aspect of the present invention relates to a method ofprolonging satiety in a subject in need thereof comprising administeringto the subject an effective amount of a ClpB protein or variant thereof,or an effective amount of a bacterium that expresses the ClpB protein orvariant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for prolonging satiety in a subject in needthereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forprolonging satiety in a subject, in particular in a subject havingnormal weight or uncomplicated overweight.

Accordingly, one aspect of the present invention relates to a method ofreducing meal size in a subject in need thereof comprising administeringto the subject an effective amount of a ClpB protein or variant thereof,or an effective amount of a bacterium that expresses the ClpB protein orvariant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for reducing meal size in a subject in needthereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forreducing meal size in a subject, in particular in a subject havingnormal weight or uncomplicated overweight.

One further aspect of the present invention relates to a method ofreducing food intake in a subject in need thereof comprisingadministering to the subject an effective amount of the ClpB protein orvariant thereof, or an effective amount of a bacterium that expressesthe ClpB protein or variant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for reducing food intake in a subject in needthereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forreducing food intake in a subject, in particular in a subject havingnormal weight or uncomplicated overweight.

One further aspect of the present invention also relates to a method ofcontrolling, in particular reducing, weight gain in a subject in needthereof comprising administering to the subject an effective amount of aClpB protein or variant thereof, or an effective amount of a bacteriumthat expresses the ClpB protein or variant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for controlling, in particular reducing, weightgain in a subject in need thereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forcontrolling, in particular reducing, weight gain in a subject, inparticular in a subject having normal weight or uncomplicatedoverweight.

One further aspect of the present invention also relates to a method ofstimulating weight loss in a subject in need thereof comprisingadministering to the subject an effective amount of a ClpB protein orvariant thereof, or an effective amount of a bacterium that expressesthe ClpB protein or variant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof for use for stimulating weight loss in a subject in needthereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forstimulating weight loss in a subject, in particular in a subject havingnormal weight or uncomplicated overweight.

One further aspect of the present invention relates to a method ofreducing fat mass on lean mass ratio in a subject in need thereof,comprising administering to the subject an effective amount of a ClpBprotein or variant thereof, or an effective amount of a bacterium thatexpresses the ClpB protein or variant thereof.

Another aspect of the present invention relates to a ClpB protein orvariant thereof, or a bacterium that expresses the ClpB protein orvariant thereof, for use for reducing fat mass on lean mass ratio in asubject in need thereof, in particular in an obese subject.

A further aspect of the present invention concerns the cosmeticnon-therapeutic use of a ClpB protein or variant thereof, or of abacterium that expresses the ClpB protein or variant thereof forreducing fat mass on lean mass ratio in a subject, in particular in asubject having normal weight or uncomplicated overweight.

As used herein, the term “subject” refers to a warm-blooded animal,preferably a human, a pet or livestock. As used herein, the terms “pet”and “livestock” include, but are not limited to, dogs, cats, guineapigs, rabbits, pigs, cattle, sheep, goats, horses and poultry. In someembodiments, the subject is a male or female subject. In someembodiments, the subject is an adult (for example, a human subject abovethe age of 18). In another embodiment, the subject is a child (forexample, a human subject below the age of 18). In some embodiments, thesubject may be a “patient”, i.e. a subject who/which is awaiting thereceipt of, or is receiving medical care or was/is/will be the object ofa medical procedure according to the methods of the present invention,or is monitored for the development of a disease.

In some embodiments, the subject is obese. “Obesity” refers herein to amedical condition wherein the subject preferably has a BMI of >30. The“BMI” or “body mass index” is defined as the subject's body mass dividedby the square of his height. The formulae universally used in medicineproduces a unit of measure of kg/m².

In some embodiments, the subject is moderately obese. A “moderatelyobese” subject refers to a subject having a BMI of between 30 and 35.

In some embodiments, the subject has a body mass index of between 18.5and 30.

In some embodiments, the subject is not obese. Typically, the non-obesesubject has a normal body weight. “Normal body weight” refers herein tobody weight resulting in a BMI of between 18.5 and 25.

In some embodiments, the subject is overweight. “Overweight” refersherein to body weight resulting in a BMI of between 25 and 30. In someembodiments, the subject is a healthy overweight or uncomplicatedoverweight subject. By “healthy overweight” or “uncomplicatedoverweight” subject is meant herein an overweight subject who does notdisplay any disease or condition directly associated with his/herweight.

In some embodiments, the subject is under a slimming diet and/or wantsto lose weight. In other embodiments, the subject is not under aslimming diet and/or does not want to lose weight.

As used herein, the term “satiety” is meant to refer to an essentiallyhomeostatic state wherein an individual feels that their cravings aresatisfied or minimized. Many physiological factors are believed to bearon an individual's satiety. For instance, gustation, or taste,olfaction, or smell, as well as a feeling of fullness of the stomach mayall contribute to whether an individual feels “satiated.” More inparticular, “satiety” is the state in which further eating is inhibitedand determines the time between meals and the amount of food consumed atthe next meal. An “enhanced feeling of satiety” or the like, this hasthe meaning of the feeling of satiety being more pronounced and/or moreprolonged compared to a control situation.

The term “satiation”, as used herein refers to the state whichterminates eating within a meal, typically occurring/observed within aperiod (e.g. 20-30 min) after the start of consuming the meal. Thus,whenever reference is made in this document to “inducing satiation” orthe like, this has the meaning of arousing the tendency of a subject tostop consuming food during a meal. The effect on satiation can bedetermined by scoring the time point of meal termination. A satiationeffect is seen if the amount of consumed calories at meal termination issignificantly less than in the controls, such as for example at least1%, 2%, 3%, 4%, 5%, 10% 20%, or more. Over a longer time period (such as1, 2, 3, 4, 5 weeks or more), one can also score the body weightreduction or the body weight change compared to a control diet. Bodyweight of a subject being administered regular amounts of the testcompositions (e.g. once daily, twice daily, or more) is preferablysignificantly controlled (reduced or less increased) compared to thecontrol subjects. As used herein, the “control subject” refers to thesubjects who were not administered with the probiotic bacterial strainof the present invention.

As used herein, the term “ClpB” has its general meaning in the art andis also known as heat shock protein F84.1 which is a member of theHsp100/ClpB family of hexameric AAA+-ATPases. ClpB has been described asan essential factor for acquired thermotolerance and for the virulenceand infectivity of several Gram-negative and Gram-positive pathogenicbacteria, such as Staphylococcus aureus, Francisella turalensis,Listeria monocytogenes, Yersinia enterocolitica, and Salmonellathyphimurium. In E. coli K12, the chaperone protein ClpB also known asheat shock protein F84.1 or htpM and is a protein of 857 amino acids.Typically, the chaperone protein ClpB comprises or consists of the aminoacid sequence of the chaperone protein ClpB from E. Coli K12 with SEQ IDNO: 1 (NCBI Reference Number: NP_417083.1, as available on Nov. 6, 2013and/or UniProtKB/Swiss-Prot Number: P63284, as available on Nov. 6,2013).

Typically, the amino acid sequence of chaperone protein ClpB comprisesor consists of an amino acid sequence 96 to 100% identical to the aminoacid sequence of SEQ ID NO: 1. Preferably, the amino acid sequence ofClpB is 96, 97, 98, 99 or 100% identical to the amino acid sequence540-550 (ARWTGIPVSR) of SEQ ID NO: 1. In the context of the presentapplication, the percentage of identity is calculated using a globalalignment (i.e. the two sequences are compared over their entirelength). Methods for comparing the identity of two or more sequences arewell known in the art. The “needle” program, which uses theNeedleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps)of two sequences when considering their entire length, may for examplebe used. The needle program is, for example, available on the ebi.ac.ukworld wide web site. The percentage of identity in accordance with theinvention is preferably calculated using the EMBOSS: needle (global)program with a “Gap Open” parameter equal to 10.0, a “Gap Extend”parameter equal to 0.5, and a Blosum62 matrix.

According to the invention the ClpB protein mimic the alpha-MSH proteinfor inducing satiation. Thus, in some embodiments, the ClpB protein ofthe present invention is recognized by an anti-alpha-MSH antibody.Typically, the antibody is a monoclonal antibody. In some embodiments,the antibody is a polyclonal antibody such as polyclonal rabbitanti-α-MSH IgG (1:1000, Peninsula Laboratories, San Carlos, Calif.,USA). The amino acid sequence of α-MSH preferably comprises or consistsof the amino acid sequence SYSMEHFRWGKPV (SEQ ID NO: 2) (Gen PeptSequence ID, PRF: 223274, as available on Dec. 2, 2013).

SEQ ID NO: 1: MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLNQEGGSVSPLL TSAGINAGQL RTDINQALNR LPQVEGTGGDVQPSQDLVRV LNLCDKLAQK RGDNFISSEL FVLAALESRGTLADILKAAG ATTANITQAI EQMRGGESVN DQGAEDQRQALKKYTIDLTE RAEQGKLDPV IGRDEEIRRT IQVLQRRTKNNPVLIGEPGV GKTAIVEGLA QRIINGEVPE GLKGRRVLALDMGALVAGAK YRGEFEERLK GVLNDLAKQE GNVILFIDELHTMVGAGKAD GAMDAGNMLK PALARGELHC VGATTLDEYRQYIEKDAALE RRFQKVFVAE PSVEDTIAIL RGLKERYELHHHVQITDPAI VAAATLSHRY IADRQLPDKA IDLIDEAASSIRMQIDSKPE ELDRLDRRII QLKLEQQALM KESDEASKKRLDMLNEELSD KERQYSELEE EWKAEKASLS GTQTIKAELEQAKIAIEQAR RVGDLARMSE LQYGKIPELE KQLEAATQLEGKTMRLLRNK VTDAEIAEVL ARWTGIPVSR MMESEREKLLRMEQELHHRV IGQNEAVDAV SNAIRRSRAG LADPNRPIGSFLFLGPTGVG KTELCKALAN FMFDSDEAMV RIDMSEFMEKHSVSRLVGAP PGYVGYEEGG YLTEAVRRRP YSVILLDEVEKAHPDVFNIL LQVLDDGRLT DGQGRTVDFR NTVVIMTSNLGSDLIQERFG ELDYAHMKEL VLGVVSHNFR PEFINRIDEVVVFHPLGEQH IASIAQIQLK RLYKRLEERG YEIHISDEALKLLSENGYDP VYGARPLKRA IQQQIENPLA QQILSGELVP GKVIRLEVNE DRIVAVQ

As used herein, “amino acids” are represented by their full name, theirthree letter code or their one letter code as well known in the art.Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G.

As used herein, the term “amino acids” includes both natural andsynthetic amino acids, and both D and L amino acids. “Standard aminoacid” or “naturally occurring amino acid” means any of the twentystandard L-amino acids commonly found in naturally occurring peptides.“Nonstandard amino acid residue” means any amino acid, other than thestandard amino acids, regardless of whether it is prepared syntheticallyor derived from a natural source. For example, naphtlylalanine can besubstituted for tryptophan to facilitate synthesis. Other syntheticamino acids that can be substituted include, but are not limited to,L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl,beta-amino acids, and isoquinolyl.

As used herein, “amino acid” also encompasses chemically modified aminoacids, including but not limited to salts, amino acid derivatives (suchas amides), and substitutions. Amino acids contained within thepolypeptides of the present invention, and particularly at the carboxy-or amino-terminus, can be modified by methylation, amidation,acetylation or substitution with other chemical groups which can changethe polypeptide's circulating half-life without adversely affectingtheir activity. Additionally, a disulfide linkage may be present orabsent in the polypeptides of the invention.

The ClpB protein or variant thereof of the invention may comprisenaturally standard amino acids or non-standard amino acids. Polypeptidemimetics include polypeptides having the following modifications:

-   -   i) polypeptides wherein one or more of the peptidyl —C(O)NR—        linkages (bonds) have been replaced by a non-peptidyl linkage        such as a —CH₂-carbamate linkage (—CH₂OC(O)NR—), a phosphonate        linkage, a —CH₂-sulfonamide (—CH₂—S(O)₂NR—) linkage, a urea        (—NHC(O)NH—) linkage, a —CH₂-secondary amine linkage, or with an        alkylated peptidyl linkage (—C(O)NR—) wherein R is C₁-C₄ alkyl;    -   ii) polypeptides wherein the N-terminus is derivatized to a        −NRR₁ group, to a —NRC(O)R group, to a —NRC(O)OR group, to a        —NRS(O)₂R group, to a —NHC(O)NHR group where R and R₁ are        hydrogen or C₁-C₄ alkyl with the proviso that R and R₁ are not        both hydrogen;    -   iii) polypeptides wherein the C terminus is derivatized to        —C(O)R₂ where R₂ is selected from the group consisting of C₁-C₄        alkoxy, and —NR₃R₄ where R₃ and R₄ are independently selected        from the group consisting of hydrogen and C₁—C4 alkyl.

In one embodiment, the ClpB protein or variant thereof comprises orconsists of amino acid sequence SEQ ID NO: 1 or variant thereof. In oneembodiment, the ClpB protein comprises or consists of amino acidsequence SEQ ID NO: 1.

In one embodiment, a “ClpB variant” as used herein comprises or consistsof a sequence presenting a sequence identity of at least 70% with aminoacid sequence SEQ ID NO: 1, preferably of at least 80%, more preferablyof at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

As used herein, the term “identity” or “identical”, when used in arelationship between the sequences of two or more amino acid sequences,refers to the degree of sequence relatedness between amino acidsequences, as determined by the number of matches between strings of twoor more amino acid residues. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). Methods for comparing theidentity of two or more sequences are well known in the art. Suchmethods include, but are not limited to, those described in Arthur M.Lesk, Computational Molecular Biology: Sources and Methods for SequenceAnalysis (New-York: Oxford University Press, 1988); Douglas W. Smith,Biocomputing: Informatics and Genome Projects (New-York: Academic Press,1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysis ofSequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar vonHeinje, Sequence Analysis in Molecular Biology: Treasure Trove orTrivial Pursuit (Academic Press, 1987); Michael Gribskov and JohnDevereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991);and Carillo et al., 1988. SIAM J. Appl. Math. 48(5):1073-1082. Preferredmethods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Computer programmethods for determining identity between two sequences include the GCGprogram package, including GAP (Devereux et al., 1984. Nucl. Acid. Res.12(1 Pt 1):387-395; Genetics Computer Group, University of WisconsinBiotechnology Center, Madison, Wis.), BLASTP, BLASTN, TBLASTN and FASTA(Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The BLASTXprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990. J. Mol. Biol.215(3):403-410). The “needle” program, which uses the Needleman-Wunschglobal alignment algorithm (Needleman S. B. and Wunsch C. D. (1970). JMol Biol 48, 443-453) to find the optimum alignment (including gaps) oftwo sequences when considering their entire length, may preferably beused. The needle program is, for example, available on the ebi.ac.ukworld wide web site. The percentage of identity in accordance with theinvention is preferably calculated using the EMBOSS: needle (global)program with a “Gap Open” parameter equal to 10.0, a “Gap Extend”parameter equal to 0.5, and a Blosum62 matrix. The well-known SmithWaterman algorithm may also be used to determine identity.

The term “variant” as used herein refers to a polypeptide that typicallydiffers from a polypeptide specifically disclosed herein in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying one or more of the above polypeptide sequences andevaluating one or more biological activities of the polypeptide asdescribed herein and/or using any of a number of techniques well knownin the art. Modifications may be made in the structure of polypeptidesand still obtain a functional molecule that encodes a variant orderivative polypeptide with desirable characteristics.

When it is desired to alter the amino acid sequence of a polypeptide tocreate an equivalent, or even an improved, variant or portion of aligand of the invention, one skilled in the art will typically changeone or more of the codons of the encoding DNA sequence. For example,certain amino acids may be substituted by other amino acids in a proteinstructure without appreciable function loss as compared to the ClpBprotein with sequence SEQ ID NO: 1.

Certain amino acid sequence substitutions can be made in a proteinsequence, and, of course, its underlying DNA coding sequence, andnevertheless obtain a protein with similar properties. It is thuscontemplated that various changes may be made in the peptide sequences,or corresponding DNA sequences that encode said peptides withoutappreciable loss of their biological utility or activity. In manyinstances, a polypeptide variant will contain one or more conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted by another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. As outlined above, amino acid substitutions aregenerally therefore based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Exemplary substitutions thattake various of the foregoing characteristics into consideration arewell known to those of skill in the art and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine. Amino acid substitutionsmay further be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includehistidine, lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) Ala, Pro,Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile,Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His.

As used herein, the term “conservative amino acid substitution” mayfurther be defined as an amino acid exchange within one of the followingfive groups:

-   -   (i) Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr, Pro, Gly;    -   (ii) Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   (iii) Polar, positively charged residues: His, Arg, Lys;    -   (iv) Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val,        Cys;    -   (v) Large, aromatic residues: Phe, Tyr, Trp.

A variant may also, or alternatively, contain non-conservative changes.In a preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more amino acids. Variants may also (or alternatively) bemodified by, for example, the deletion or addition of amino acids thathave minimal influence on the immunogenicity, secondary structure andhydropathic nature of the polypeptide.

In one embodiment, a variant of SEQ ID NO: 1 is capable of inducingsatiation, prolonging satiety, reducing food intake, controlling weightgain, stimulating weight loss and/or reducing fat mass on lean massratio in a subject in need thereof with an efficiency at leastequivalent to the one of SEQ ID NO: 1.

In one embodiment, a variant of SEQ ID NO: 1 comprises conservativeamino acid substitutions as compared to the sequence of SEQ ID NO: 1,such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativeamino acid substitutions.

In another embodiment, a variant of SEQ ID NO: 1 is a polypeptidewherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from thesequence of SEQ ID NO: 1 is/are absent, or substituted by any aminoacid, or wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids(either contiguous or not) is/are added.

In one embodiment of the invention, the ClpB protein or variant thereofas described here above are modified by means well-known in the art, forinstance by the addition of one or more functional group such as aphosphate, acetate, lipid or carbohydrate group, and/or by the additionof one or more protecting group.

For example, the ClpB protein or variant thereof can be modified by theaddition of one or more functional groups such as phosphate, acetate, orvarious lipids and carbohydrates. The ClpB protein or variant thereofcan also exist as polypeptide derivatives.

The term “polypeptide derivative” refers to compound having an aminogroup (—NH—), and more particularly, a peptide bond. Polypeptides may beregarded as substituted amides. Like the amide group, the peptide bondshows a high degree of resonance stabilization. The C—N single bond inthe peptide linkage has typically about 40 percent double-bond characterand the C═O double bond about 40 percent single-bond character.“Protecting groups” are those groups that prevent undesirable reactions(such as proteolysis) involving unprotected functional groups. Specificexamples of amino protecting groups include formyl; trifluoroacetyl;benzyloxycarbonyl; substituted benzyloxycarbonyl such as (ortho- orpara-) chlorobenzyloxycarbonyl and (ortho- or para-)bromobenzyloxycarbonyl; and aliphatic oxycarbonyl such ast-butoxycarbonyl and t-amiloxycarbonyl. The carboxyl groups of aminoacids can be protected through conversion into ester groups. The estergroups include benzyl esters, substituted benzyl esters such asmethoxybenzyl ester; alkyl esters such as cyclohexyl ester, cycloheptylester or t-butyl ester. The guanidino moiety may be protected by nitro;or arylsulfonyl such as tosyl, methoxybenzensulfonyl ormesitylenesulfonyl, even though it does not need a protecting group. Theprotecting groups of imidazole include tosyl, benzyl and dinitrophenyl.The indole group of tryptophan may be protected by formyl or may not beprotected.

The modification of the ClpB protein or variant thereof aims inparticular to improve their life time in vivo. One type of modificationis the addition to the N or C termini of the ClpB protein or variantthereof of polyethylene glycol (PEG). PEG is known by the person skilledin the art to have many properties that make it an ideal carrier forpolypeptides such as high water solubility, high mobility in solutionand low immunogenicity. This modification also protects the polypeptidesfrom exopeptidases and therefore increases their overall stability invivo.

The other modifications used to prevent degradation of the polypeptidesby endopeptidases or exopeptidases include N-terminal modifications suchas acetylation or glycosylation, C-terminal modifications such asamidation and use of unnatural amino acids (β-amino andα-trifluoromethyl amino acids) at particular sites within thepolypeptides.

Another alternative to increase polypeptide molecular size is thegenetic fusion of the polypeptides to the Fc domain of humanimmunoglobulin (including, for example, IgA, IgM and IgG) or the fusionof the polypeptides to albumin.

The ClpB protein or variant thereof described herein can be producedsynthetically by chemical synthesis or enzymatic synthesis as it is wellknown in the art. Alternatively, nucleotide sequences encoding thepolypeptides of the invention can be introduced into a proteinexpression vector and produced in a suitable host organism (e.g.,bacteria, insect cells, etc.), then purified. In one embodiment, theClpB protein or variant thereof is obtained by a cloning method, suchas, for example, using any production system known in the art, such as,for example, E. coli, yeast, baculovirus-insect cell, or mammalian cellssuch as HEK or CHO expression system.

An additional polypeptide (“tag”) can be added on for the purpose ofpurifying or identifying or purifying the polypeptides. Protein tagsmake it possible, for example, for the polypeptides to be adsorbed, withhigh affinity, to a matrix, and for the matrix then to be washedstringently with suitable buffers without the complex being eluted toany significant extent, and for the adsorbed complex subsequently to beeluted selectively.

Examples of protein tags which are known to the skilled person are a(His)₆ tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathionetransferase (GST) tag, intein having an affinity chitin-binding tag ormaltose-binding protein (MBP) tag. These protein tags can be locatedN-terminally, C-terminally and/or internally.

In some embodiments, the ClpB protein is administered to the subject inthe form of a pharmaceutical composition. In some embodiments, the ClpBprotein is combined with pharmaceutically acceptable excipients, andoptionally sustained-release matrices, such as biodegradable polymers,to form pharmaceutical compositions.

The term “pharmaceutically” or “pharmaceutically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a mammal,especially a human, as appropriate. A pharmaceutically acceptablecarrier or excipient refers to a non-toxic solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. In the pharmaceutical compositions of the present invention, theactive principle, alone or in combination with another active principle,can be administered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.Suitable unit administration forms comprise oral-route forms such astablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, aerosols,implants, subcutaneous, transdermal, topical, intraperitoneal,intramuscular, intravenous, subdermal, transdermal, intrathecal andintranasal administration forms and rectal administration fottns.Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The activeingredient can be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. The carrier can alsobe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetables oils. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin. Sterile injectable solutions are prepared byincorporating the active polypeptides in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. Upon formulation, solutions will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective. The formulations are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but drug release capsules and thelike can also be employed. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 mL of isotonic NaCl solution and either added to 1000 mLof hypodermoclysis fluid or injected at the proposed site of infusion.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

As used herein, the expression “bacterium that expresses the ClpB”refers to a bacterium expressing or over-expressing the chaperoneprotein ClpB or variant thereof, as defined above or a polypeptidecomprising or consisting of an amino acid sequence 96 to 100% identicalto the amino acid sequence of SEQ ID NO: 1, more preferably 96, 97, 98,99 or 100% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the bacterium that expresses the ClpB protein orvariant thereof is a food grade bacterium.

In some embodiments, the bacterium that expresses the ClpB protein orvariant thereof is a probiotic bacterial strain.

As used herein, the term “probiotic” is meant to designate livemicroorganisms which, when they are integrated in a sufficient amount,exert a positive effect on health, comfort and wellness beyondtraditional nutritional effects. Probiotic microorganisms have beendefined as “Live microorganisms which when administered in adequateamounts confer a health benefit on the host” (FAO/WHO 2001). As usedherein, the expression “probiotic bacterial strain” denotes a bacterialstrain that has a beneficial effect on the health and well-being of thehost.

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, of the present invention is a viable bacterial strain,in particular a viable probiotic bacterial strain. The expression“viable bacterial strain” means a microorganism which is metabolicallyactive and that is able to colonize the gastro-intestinal tract of thesubject.

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, of the present invention is a non-viable bacterialstrain, in particular probiotic bacterial strain, consisting of amixture of bacterial fragments. In some embodiments, the mixture ofbacterial fragments of the present invention consists of proteins fromthe bacterial strain.

In some embodiments, the probiotic bacterial stain of the presentinvention is selected from food grade bacteria. “Food grade bacteria”means bacteria that are used and generally regarded as safe for use infood.

The bacterial strain may be a naturally occurring bacterial strain or itmay be a genetically engineered bacterial strain.

In some embodiments, the bacterial strain, in particular probioticbacterial strain, of the present invention is a bacterium whichconstitutively expresses the ClpB protein or variant thereof.

In some embodiments, the ClpB protein or variant thereof isoverexpressed in the bacterium. Generally, a protein expression is“upregulated” or a protein is “over-expressed” when it is expressed orproduced in an amount or yield that is higher than a given base-lineyield that occurs in nature at standard conditions. Over-expression of aprotein can be achieved, for example, by altering any one or more of:(a) the growth or living conditions of the host cells; (b) thepolynucleotide encoding the protein; (c) the promoter used to controlexpression of the polynucleotide and its copy number in the cell;

and (d) the host cells themselves.

In some embodiments, the bacterium was subjected to stress conditions sothat the expression of the ClpB protein or variant thereof is upregulated in the bacterium. Stress may be selected from the groupconsisting of an exposure to heat, temperature changes, mechanicalstress, or long term storage, low moisture storage and/or freeze dryingor spray drying.

In some embodiments, the bacterium was subjected to nutrient supply forat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20times. Typically, the nutrients are supplied by the means of a culturemedium that is convenient for the growth of the bacterium, e.g.Muller-Hinton as described in the examples. In sonic embodiments, thebacterium is isolated at the stage of the stationary phase that followssaid repeated nutrient supply, since during this phase the concentrationof the ClpB protein or variant thereof is maximal.

In some embodiments, the bacterium comprises at least one point mutationso that the expression of the ClpB protein or variant thereof is upregulated. The term “point mutation” as used herein means a nucleic acidsubstitution and/or deletion. Alternatively or simultaneously, the atleast one mutation is located within the regulatory DNA sequences of theClpB gene, e.g., in the transcriptional and translational controlsequences, it is preferred that this mutation modulates the expressionof the protein. Mutations within the regulatory DNA sequences may serveto upregulate the expression of the protein.

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, of the present invention is a bacterium which has beengenetically engineered for expressing the ClpB protein or variantthereof. Typically, the bacterial strain was transformed with a nucleicacid encoding for the ClpB protein or variant thereof. The term“transformation” means the introduction of a “foreign” (i.e. extrinsicor extracellular) gene, DNA or RNA sequence to a host cell, so that thehost cell will express the introduced gene or sequence to produce adesired substance, typically a protein or enzyme coded by the introducedgene or sequence. A host cell that receives and expresses introduced DNAor RNA has been “transformed”. The nucleic acid may remainextra-chromosomal upon transformation of a parental microorganism or maybe adapted for integration into the genome of the microorganism.Accordingly, the nucleic acid may include additional nucleotidesequences adapted to assist integration (for example, a region whichallows for homologous recombination and targeted integration into thehost genome) or stable expression and replication of an extrachromosomalconstruct (for example, origin of replication, promoter and otherregulatory sequences). In some embodiments, the nucleic acid is nucleicacid construct or vector. In some embodiments, the nucleic acidconstruct or vector is an expression construct or vector, however otherconstructs and vectors, such as those used for cloning are encompassedby the invention. In some embodiments, the expression construct orvector is a plasmid. Typically, the expression construct/vector furthercomprises a promoter, as herein before described. In some embodiments,the promoter allows for constitutive expression of the genes under itscontrol. However, inducible promoters may also be employed. It will beappreciated that an expression construct/vector of the present inventionmay contain any number of regulatory elements in addition to thepromoter as well as additional genes suitable for expression of the ClpBprotein or variant thereof if desired. Methods for transformingbacterial cell with extracellular nucleic acids are well known in theart.

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, is a gram-negative strain.

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, is a member of the family of Enterobacteriaceae. Inparticular, the bacterial strain is a non-pathogenic member of thefamily of Enterobacteriaceae.

Interestingly, the inventors showed that the ClpB protein of allEnterobacteriaceae bacteria displayed 100% identity with each other. Insome embodiments, the bacterial strain, in particular the probioticbacterial strain, is an E. coli strain. In some embodiments, E. colistrains, in particular probiotic E. coli strains, for use according tothe teachings of present invention include non-pathogenic E. colistrains which exert probiotic activity. Example of probiotic E. colistrain is the probiotic Escherichia coil strain BU-230-98, ATCC DepositNo. 202226 (DSM 12799), which is an isolate of the known, commerciallyavailable, probiotic Escherichia coli strain M-17. Example of anon-pathogenic E. coli strain is E. coli Nissle 1917. An example of E.coli strain which was not known as probiotic is the laboratory E. colistrain K12.

In other embodiments, the bacterial strain is a Hafnia alvei strain,such as the Hafnia alvei strain AF036 commercialized by Bioprox Company.In still other embodiments, the bacterial strain is a Proteus vulgarisstrain.

In still other embodiments, a combination of bacterial strainsexpressing the ClpB protein or variant thereof is used.

Typically, the bacterial strain, in particular the probiotic bacterialstrain, of the present invention is produced with any appropriateculture medium well known in the art. Various fermentation media aresuitable according to the invention, such as (but not limited to) e.g.firstly an industrial medium, in which the strain(s) is/are grown, andthat is used as is or after concentration (e.g. drying) or afteraddition to another food base or product. Alternatively, bacterialcells, or bacterial cells with medium (e.g. the fermentation broth), orfractions of such cell comprising medium (i.e. medium with saidbacterial strain/s) may be used. The cells or the cell comprising mediumcomprise live or viable bacterial cells and/or dead or non-viablebacterial cells of the strain(s). The medium may thus be treated by, butnot limited to, heating or sonication. Also, lyophilized or frozen,bacteria and/or cell-free media (which may be concentrated) areencompassed in the methods for preparing the bacterial strain, inparticular the probiotic bacterial strain, of the present invention.

In a particular embodiment, the bacterial strain of the invention islyophilized, then preferably resuspended before being administered.

Typically, the bacterial strain, in particular the probiotic bacterialstrain, of the present invention is administered to the subject byingestion (i.e. oral route).

In some embodiments, the bacterial strain, in particular the probioticbacterial strain, of the present invention is encapsulated in order tobe protected against the stomach. Accordingly, in some embodiments thebacterial strain, in particular the probiotic bacterial strain, of thepresent invention is formulated in compositions in an encapsulated formso as significantly to improve their survival time. In such a case, thepresence of a capsule may in particular delay or prevent the degradationof the microorganism in the gastrointestinal tract. It will beappreciated that the compositions of the present embodiments can beencapsulated into an enterically-coated, time-released capsule ortablet. The enteric coating allows the capsule/tablet to remain intact(i.e., undissolved) as it passes through the gastrointestinal tract,until such time as it reaches the small intestine. Methods ofencapsulating live bacterial cells are well known in the art (see, e.g.,U.S. patents to General Mills Inc. such as U.S. Pat. No. 6,723,358). Forexample, micro-encapsulation with alginate and Hi-Maize™ starch followedby freeze-drying has been proved successful in prolonging shelf-life ofbacterial cells in dairy products [see, e.g., Kailasapathy K. (2002)Curr Issues Intest Microbiol 3(2), 39-48]. Alternatively, encapsulationcan be done with glucornannane fibers such as those extracted fromAmorphophallus konjac. Alternatively, entrapment of viable bacteria insesame oil emulsions may also be used [see, e.g., Hou R. C., Lin M. Y.,Wang M. M., Tzen J. T. (2003) J Dairy Sci 86, 424-428]. In someembodiments, agents for enteric coatings are preferably methacrylicacid-alkyl acrylate copolymers, such as Eudragit® polymers.Poly(meth)acrylates have proven particularly suitable as coatingmaterials. EUDRAGIT® is the trade name for copolymers derived fromesters of acrylic and methacrylic acid, whose properties are determinedby functional groups. The individual EUDRAGIT® grades differ in theirproportion of neutral, alkaline or acid groups and thus in terms ofphysicochemical properties. The skillful use and combination ofdifferent EUDRAGIT® polymers offers ideal solutions for controlled drugrelease in various pharmaceutical and technical applications. EUDRAGIT®provides functional films for sustained-release tablet and pelletcoatings. The polymers are described in international pharmacopeias suchas Ph.Eur., USP/NF, DMF and JPE. EUDRAGIT® polymers can provide thefollowing possibilities for controlled drug release: gastrointestinaltract targeting (gastroresistance, release in the colon), protectivecoatings (taste and odor masking, protection against moisture) anddelayed drug release (sustained-release formulations). EUDRAGIT®polymers are available in a wide range of different concentrations andphysical forms, including aqueous solutions, aqueous dispersion, organicsolutions, and solid substances. The pharmaceutical properties ofEUDRAGIT® polymers are determined by the chemical properties of theirfunctional groups. A distinction is made between:

-   -   poly(meth)acrylates, soluble in digestive fluids (by salt        formation) EUDRAGIT® L (Methacrylic acid copolymer), S        (Methacrylic acid copolymer), FS and E (basic butylated        methacrylate copolymer) polymers with acidic or alkaline groups        enable pH-dependent release of the active ingredient.        Applications: from simple taste masking via resistance solely to        gastric fluid, to controlled drug release in all sections of the        intestine.    -   poly(meth)acrylates, insoluble in digestive fluids: EUDRAGIT® RL        and RS (ammonio methacrylate copolymers) polymers with alkaline        and EUDRAGIT® NE polymers with neutral groups enable controlled        time release of the active by pH-independent swelling.

Enteric EUDRAGIT® coatings provide protection against drug release inthe stomach and enable controlled release in the intestine. The dominantcriterion for release is the pH-dependent dissolution of the coating,which takes place in a certain section of the intestine (pH 5 to over 7)rather than in the stomach (pH 1-5). For these applications, anionicEUDRAGIT® grades containing carboxyl groups can be mixed with eachother. This makes it possible to finely adjust the dissolution pH, andthus to define the drug release site in the intestine. EUDRAGIT® L and Sgrades are suitable for enteric coatings.

EUDRAGIT® FS 30 D (aqueous dispersion of an anionic copolymer based onmethyl acrylate, methyl methacrylate and methacrylic acid) isspecifically used for controlled release in the colon.

Typically, the bacterial strain, in particular the probiotic bacterialstrain, of the present invention is administered to the subject in theform of a food composition. Accordingly, one further aspect of thepresent invention relates to a food composition comprising an amount ofthe bacterial strain, in particular the probiotic bacterial strain, ofthe present invention.

In some embodiments, the food composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention is selected from complete food compositions, food supplements,nutraceutical compositions, and the like. The composition of the presentinvention may be used as a food ingredient and/or feed ingredient.

The food ingredient may be in the form of a solution or as a solid,depending on the use and/or the mode of application and/or the mode ofadministration.

The bacterial strain, in particular the probiotic bacterial strain, ofthe present invention is typically added at any time during theproduction process of the composition, e.g. they may be added to a foodbase at the beginning of the production process or they may be added tothe final food product.

“Food” refers to liquid (i.e. drink), solid or semi-solid dieteticcompositions, especially total food compositions (food-replacement),which do not require additional nutrient intake or food supplementcompositions. Food supplement compositions do not completely replacenutrient intake by other means. Food and food supplement compositionsare for example fermented dairy products or dairy-based products, whichare preferably administered or ingested orally one or more times daily.Fermented dairy products can be made directly using the bacteriaaccording to the invention in the production process, e.g. by additionto the food base, using methods known per se. In such methods, thestrain(s) of the invention may be used in addition to the micro-organismusually used, and/or may replace one or more or part of themicro-organism usually used. For example, in the preparation offermented dairy products such as yoghurt or yoghurt-based drinks, abacterium of the invention may be added to or used as part of a starterculture or may be suitably added during such a fermentation. Optionallythe bacteria may be inactivated or killed later in the productionprocess. Fermented dairy products include milk-based products, such as(but not limited to) deserts, yoghurt, yoghurt drinks, quark, kefir,fermented milk-based drinks, buttermilk, cheeses, dressings, low fatspreads, fresh cheese, soy-based drinks, ice cream, etc. Alternatively,food and/or food supplement compositions may be non-dairy or dairynon-fermented products (e.g. strains or cell-free medium innon-fermented milk or in another food medium). In some embodiments, theprobiotic bacterial strain of the present invention is encapsulated anddispersed in a food (e.g. in milk) or non-food medium. Non-fermenteddairy products may include ice cream, nutritional bars and dressings,and the like. Non-dairy products may include powdered beverages andnutritional bars, and the like. The products may be made using knownmethods, such as adding an effective amount of the strain(s) and/orcell-free culture medium to a food base, such as skimmed milk or milk ora milk-based composition and fermentation as known. Other food bases towhich the (compositions comprising the) bacterial cells and/or cell-freeculture medium may be added are meat, meat replacers or plant bases.

The composition that comprises the bacterial strain, in particular theprobiotic bacterial strain, of the present invention may be solid,semi-solid or liquid. It may be in the form of a food product or foodsupplement, e.g. in the fot in of tablets, gels, powders, capsules,drinks, bars, etc. For example, the composition may be in the form of apowder packed in a sachet which can be dissolved in water, fruit juice,milk or another beverage.

As used herein, the term “food ingredient” or “feed ingredient” includesa formulation which is or can be added to functional foods or foodstuffsas a nutritional supplement.

By “nutritional food” or “nutraceutical” or “functional” food, is meanta foodstuff which contains ingredients having beneficial effects forhealth or capable of improving physiological functions.

By “food supplement”, is meant a foodstuff having the purpose ofcompleting normal food diet. A food supplement is a concentrated sourceof nutrients or other substances having a nutritional or physiologicaleffect, when they are taken alone or as a combination in small amounts.

According to the invention, “functional food” summarizes foodstuff andcorresponding products lately developed to which importance isattributed not only due to them being valuable as to nutrition and tastebut due to particular ingredient substances. According to the invention,the middle- or long-term maintenance and promotion of health are ofimportance. In this context, non-therapeutic uses are preferred. Theterms “nutriceuticals”, “foodsceuticals” and “designer foods”, whichalso represent embodiments of the invention, are used as synonyms,partly, however, also in a differentiated way. The preventive aspect andthe promotion of health as well as the food character of the productsare, however, best made clear by the term functional food. In manycases, these relate to products accumulated by assortment and selection(as is also the case in the present invention), purification,concentration, increasingly also by addition. Isolated effectivesubstances, in particular in form of tablets or pills, are not included.Although there is no legal definition of a functional food, most of theparties with an interest in this area agree that they are foods marketedas having specific health effects beyond basic nutritional effects.Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional e.g. medical or physiologicalbenefit other than a purely nutritional effect.

In some embodiments, the drink is a functional drink or a therapeuticdrink, a thirst-quencher or an ordinary drink. By way of example, thecomposition of the present invention can be used as an ingredient tosoft drinks, a fruit juice or a beverage comprising whey protein, healthteas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurtand drinking yoghurt, cheese, ice cream, water ices and desserts,confectionery, biscuits cakes and cake mixes, snack foods, balancedfoods and drinks, fruit fillings, care glaze, chocolate bakery filling,cheese cake flavored filling, fruit flavored cake filling, cake anddoughnut icing, instant bakery filling creams, fillings for cookies,ready-to-use bakery filling, reduced calorie filling, adult nutritionalbeverage, acidified soy/juice beverage, aseptic/retorted chocolatedrink, bar mixes, beverage powders, calcium fortified soy/plain andchocolate milk, calcium fortified coffee beverage.

The composition can further be used as an ingredient in food productssuch as American cheese sauce, anti-caking agent for grated & shreddedcheese, chip dip, cream cheese, dry blended whip topping fat free sourcream, freeze/thaw dairy whipping cream, freeze/thaw stable whippedtopping, low fat and light natural cheddar cheese, low fat Swiss styleyoghurt, aerated frozen desserts, hard pack ice cream, label friendly,improved economics & indulgence of hard pack ice cream, low fat icecream: soft serve, barbecue sauce, cheese dip sauce, cottage cheesedressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauceand others.

In some embodiments, the composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention is used with yoghurt production, such as fermented yoghurtdrink, yoghurt, drinking yoghurt, cheese, fermented cream, milk baseddesserts and others. Suitably, the composition can be further used as aningredient in one or more of cheese applications, meat applications, orapplications comprising protective cultures.

In some embodiments, the food composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention is suitable for preparing meal replacement product. As usedherein, the term “meal replacement product” unless otherwise specified,includes any nutritional product containing protein, carbohydrate,lipid, vitamins and minerals, the combination of which is then suitableas a sole or primary nutrition source for a meal. Typically, the mealreplacement product comprises at least one carbohydrate source, at leastone lipid source and/or at least one protein source. As protein source,any suitable dietary protein may be used, for example animal proteins(such as milk proteins, meat proteins and egg proteins); vegetableproteins (such as soy protein, wheat protein, rice protein, and peaprotein); mixtures of free amino acids; or combinations thereof. Milkproteins such as casein and whey, and soy proteins are particularlypreferred. The proteins may be intact or hydrolyzed or a mixture ofintact and hydrolyzed proteins. It may be desirable to supply partiallyhydrolyzed proteins (degree of hydrolysis between 2 and 20%), forexample for animals believed to be at risk of developing cows' milkallergy. If hydrolyzed proteins are required, the hydrolysis process maybe carried out as desired and as is known in the art. For example, awhey protein hydrolysate may be prepared by enzymatically hydrolyzingthe whey fraction in one or more steps. If the whey fraction used as thestarting material is substantially lactose free, it is found that theprotein suffers much less lysine blockage during the hydrolysis process.This enables the extent of lysine blockage to be reduced from about 15%by weight of total lysine to less than about 10% by weight of lysine;for example about 7% by weight of lysine which greatly improves thenutritional quality of the protein source. If the composition includes afat source, the fat source preferably provides 5% to 40% of the energyof the composition; for example 20% to 30% of the energy. A suitable fatprofile may be obtained using a blend of canola oil, corn oil andhigh-oleic acid sunflower oil. The source of carbohydrates preferablyprovides 40% to 80% of the energy of the composition. Any suitablecarbohydrate may be used, for example sucrose, lactose, glucose,fructose, corn syrup solids, maltodextrins, and mixtures thereof.Typically, substituting one daily meal by an energy restricted diet witha meal replacement contributes to the maintenance of weight after weightloss.

The food composition that comprises the bacterial strain, in particularthe probiotic bacterial strain, of the present invention typicallycomprises carriers or vehicles. “Carriers” or “vehicles” mean materialssuitable for administration and include any such material known in theart such as, for example, any liquid, gel, solvent, liquid diluent,solubilizer, or the like, which is non-toxic and which does not interactwith any components, in particular with the bacterial strain, of thecomposition in a deleterious manner. Examples of nutritionallyacceptable carriers include, for example, water, salt solutions,alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethyleneglycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose,magnesium stearate, talc, surfactants, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, petroethralfatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, andthe like.

In some embodiments, the food composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention comprises an amount of dietary fibers. Dietary fiber passesthrough the small intestine undigested by enzymes and functions as anatural bulking agent and laxative. Dietary fiber may be soluble orinsoluble and in general a blend of the two types is preferred. Suitablesources of dietary fiber include soy, pea, oat, pectin, guar gum, gumArabic, fructooligosaccharides, galacto-oligosaccharides, sialyl-lactoseand oligosaccharides derived from animal milks. In some embodiments, thedietary fiber is selected among mannans. Mannans (such as glucomannansand galactomannans), such as guar gum, locust bean gum, konjac, andxanthan gum, are present in some plant cell walls. The glucomannans aregenerally comprised of (1-4)-β-linked glucose and mannose units, whilethe galactomannans are generally comprised of a (1-4)-β-mannan backbonesubstituted with single units of (1-6)-α-galactose. Many endospermiclegumes, such as guar and locust bean, contain galactomannans in theendosperm during seed development. Glucomannans have also been found asa minor component of cereal grains.

In some embodiments, the food composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention contains minerals and micronutrients such as trace elementsand vitamins in accordance with the recommendations of Government bodiessuch as the USRDA. For example, the composition may contain per dailydose one or more of the following micronutrients in the ranges given:300 to 500 mg calcium, 50 to 100 mg magnesium, 150 to 250 mg phosphorus,5 to 20 mg iron, 1 to 7 mg zinc, 0.1 to 0.3 mg copper, 50 to 200 μgiodine, 5 to 15 μg selenium, 1000 to 3000 μg beta carotene, 10 to 80 mgVitamin C, 1 to 2 mg Vitamin B1, 0.5 to 1.5 mg Vitamin B6, 0.5 to 2 mgVitamin B2, 5 to 18 mg niacin, 0.5 to 2.0 μg Vitamin B12, 100 to 800 μgfolic acid, 30 to 70 μg biotin, 1 to 5 μg Vitamin D, 3 to 10 μg VitaminE.

In some embodiments, the composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention contains emulsifiers. Examples of food grade emulsifierstypically include diacetyl tartaric acid esters of mono- anddi-glycerides, lecithin and mono- and di-glycerides. Similarly, suitablesalts and stabilizers may be included.

In some embodiments, the food composition that comprises the probioticbacterial strain of the present invention contains at least oneprebiotic. “Prebiotic” means food substances intended to promote thegrowth of the probiotic bacterial strain of the present invention in theintestines. The prebiotic may be selected from the group consisting ofoligosaccharides and optionally contains fructose, galactose, mannose,soy and/or inulin; and/or dietary fibers.

In some embodiments, the composition that comprises the bacterialstrain, in particular the probiotic bacterial strain, of the presentinvention contains protective hydrocolloids (such as gums, proteins,modified starches), binders, film forming agents, encapsulatingagents/materials, wall/shell materials, matrix compounds, coatings,emulsifiers, surface active agents, solubilizing agents (oils, fats,waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds,dispersing agents, wetting agents, processing aids (solvents), flowingagents, taste masking agents, weighting agents, jellifying agents, gelforming agents, antioxidants and antimicrobials. The composition mayalso contain conventional pharmaceutical additives and adjuvants,excipients and diluents, including, but not limited to, water, gelatinof any origin, vegetable gums, ligninsulfonate, talc, sugars, starch,gum arabic, vegetable oils, polyalkylene glycols, flavoring agents,preservatives, stabilizers, emulsifying agents, buffers, lubricants,colorants, wetting agents, fillers, and the like. In all cases, suchfurther components will be selected having regard to their suitabilityfor the intended recipient.

In some embodiments, the administration of the ClpB protein or variantthereof, or of the bacterial strain, in particular the probioticbacterial strain, that expresses the ClpB protein or variant thereof, isrepeated, for example, 2 to 3 times a day, for one day or more andgenerally for a sustained period of at least 4 days, or even 4 to 15weeks, with, where appropriate, one or more periods of interruption. Insome embodiments, the ClpB protein or variant thereof or the bacterialstrain that expresses the ClpB protein or variant thereof isadministered simultaneously or sequentially with one meal of thesubject. In some embodiments, the ClpB protein or variant thereof or thebacterial strain that expresses the ClpB protein or variant thereof, isadministered prior to the meal of the subject.

As used herein, the term “effective amount” refers to a quantitysufficient of ClpB protein or variant thereof or of bacteria expressingthe ClpB protein or variant thereof, to achieve the beneficial effect(e.g. stimulating satiety, prolonging satiation, reducing food intake,controlling, in particular reducing, weight gain, stimulating weightloss, and/or reducing fat mass on lean mass ratio). In the context ofthe present invention, the amount of a ClpB protein or variant thereofor of bacteria expressing the ClpB protein or variant thereof,administered to the subject will depend on the characteristics of theindividual, such as general health, age, sex, body weight. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors. For example, when the ClpB protein or variant thereofis administered to the subject in the form a probiotic, the strain ofthe present invention shall be able to generate a colony which issufficient to generate a beneficial effect on the subject. If thebacterial strain, in particular the probiotic bacterial strain, isadministered in the form of a food product, it typically may comprisebetween 10³ and 10¹² cfu of the bacterial strain, in particular theprobiotic bacterial strain, of the present invention per g of the dryweight of the food composition.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effects of daily intragastric delivery of E. coli K12 wild type(WT) or E. coli deleted for ClpB gene (ΔClpB) in normal C57B16 adultmice on body weight (A, E), 24 h food intake (B, F) and meal pattern (C,D, G, H) analyzed at the beginning and the end of 3 weeks of gavage.Control mice (Ctr) did not receive any gavage. Meal pattern was measuredas meal size (C, G), corresponding to an average amount of food eatenduring a single meal, and as meal frequency (D, H), corresponding to anumber of meals per 24 h separated by at least 5 min. Decreased mealsize reflects faster satiation and decreased meal frequency reflectslonger satiety. A: Two-way repeated measurements ANOVA, Bonferronipost-test *p<0.05. B, D, G, H: ANOVA, Tukey's post-tests, *p<0.05,**p<0.01, ***p<0.001. E: Student's t-test, *p<0.05.

FIG. 2: Effect of ClpB on electrical activity of ARC POMC neurons.Action potential frequency expressed in percent of change versus basallevel; mean+SEM. Bonferroni post-tests *p<0.05, **p<0.01.

FIG. 3: Body weight dynamics in obese ob/ob mice before and during (Days0-21) intragastric gavage with E. coli K12, E. coli K12 ΔClpB, both inMueller-Hilton (MH) medium, or with MH medium only, as a control (Ctr).2-way ANOVA, Effect of treatment: p=0.01, Bonferroni post-tests Ctr. vs.E. coli K12, * p<0.05 and **p<0.01. Mean+SEM.

FIG. 4: Percentage of mean body weight change (from the day ofrandomization=100%) in obese ob/ob mice after 3 weeks of intragastricgavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both inMueller-Hilton (MH) medium, or with the MH medium only, as a control(Ctr., n=7). ANOVA p=0.01, Tukey's post-tests * p<0.05. Mean+SEM.

FIG. 5: Fat content in obese ob/ob mice measured by EchoMRI after 3weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB(n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only,as a control (Ctr., n=7). ANOVA p=0.005, Tukey's post-test **p<0.01.Mean±SEM.

FIG. 6: Lean to fat mass ratios in obese ob/ob mice measured by EchoMRIafter 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH mediumonly, as a control (Ctr., n=7). ANOVA p=0.05, Student's t-test *p<0.05.Mean+SEM.

FIG. 7: Mean daily food intake in obese ob/ob mice during 3 weeks ofintragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8),both in Mueller-Hilton (MH) medium, or with the MH medium only, as acontrol (Ctr., n=7). ANOVA p<0.0001, Tukey's post-test ***p<0.001.Mean+SEM.

FIG. 8: Mean daily meal number in obese ob/ob mice during 3 weeks ofintragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8),both in Mueller-Hilton (MH) medium, or with the MH medium only, as acontrol (Ctr., n=7). ANOVA p<0.0001, Tukey's post-test ***p<0.001.Mean±SEM.

FIG. 9: Daily body weight gain in obese ob/ob mice during intragastricgavage with E. coli K12, E. coli Niessle 1917, E. coli Niessle 1917lyophilized (lyo), all in Mueller-Hilton (MH) medium, or with MH mediumonly, as a control (Ctr). 2-way ANOVA, p=0.02, Bonferroni post-tests a,Ctr. vs. E. coli Niessle 1917 lyo and b, Ctr. vs. E. coli Niessle1917, * p<0.05 and **p<0.01. Mean±SEM.

FIG. 10: Percentage of mean body weight change (from the day ofrandomization=100%) in obese ob/ob mice after 2 weeks of intragastricgavage with E. coli K12, E. coli Niessle 1917, E. coli Niessle 1917lyophilized (lyo), all in Mueller-Hilton (MH) medium, or with MH mediumonly, as a control (Ctr). Kruskal-Wallis p<0.01, Dunn's post-test*p<0.05, Mann-Whitney test ##p<0.01. Mean±SEM.

FIG. 11: Fat content in obese ob/ob mice measured by EchoMRI after 2weeks of intragastric gavage with E. coli K12, E. coli Niessle 1917, E.coli Niessle 1917 lyophilized (lyo), all in Mueller-Hilton (MH) medium,or with MH medium only, as a control (Ctr). Student's t-test **p<0.01.Mean±SEM.

FIG. 12: Effect of a direct intracerebroventricular injection ofdifferent doses of ClpB in food intake in rats.

FIG. 13: Body weight gain (in g) in obese ob/ob mice after 17 days ofintragastric gavage with E. coli Niessle 1917 (Nissle) (n=15), E. coliK12 (K12 Δ-ClpB) (n=15) or Hafnia alvei AF036 (Hafnia) (n=15), all inMueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl)(n=12).

FIG. 14: Cumulative food intake (in g) in obese ob/ob mice after 17 daysof intragastric gavage with E. coli Niessle 1917 (Nissle) (n=15), E.coli K12 (K12 Δ-ClpB) (n=15) or Hafnia alvei AF036 (Hafnia) (n=15), allin Mueller-Hilton (MH) medium, or with MH medium only, as a control(Ctrl) (n=12).

FIG. 15: Fat and lean mass gain in obese ob/ob mice measured by EchoMRIafter 17 days of intragastric gavage with E. coli Niessle 1917 (Nissle)(n=15), E. coli K12 (K12 ΔClpB) (n=15) or Hafnia alvei AF036 (H. alveri)(n=15), all in Mueller-Hilton (MH) medium, or with MH medium only, as acontrol (Ctrl) (n=12). A. Fat mass gain (in g). B. Lean mass gain (ing). C. Lean-to-fat mass ratios.

FIG. 16: Relative hormone-sensitive lipase protein (pHSL) expressionrate (against actine expression rate as a standard) in obese ob/ob miceafter 17 days of intragastric gavage with E. coli Niessle 1917 (E. coliNissle) (n=15), E. coli K12 (E. coli K12) (n=15) or Hafnia alvei AF036(Hafnia alvei) (n=15), all in Mueller-Hilton (MH) medium, or with MHmedium only, as a control (Ctrl) (n=12). HSL and actine expression rateswere measured by western blot.

FIG. 17: High fat diet validation. A. Body weight (in g) in mice fedwith a high fat/high carbs diet (HFD) (n=48) and in mice fed with acontrol diet (Ctrl) (n=8). B. Fat mass (in g). C. Lean mass (in g).

FIG. 18: Reduction in body weight gain from D0 in high fat diet(HFD)-induced obese mice after 14 days of intragastric gavage with E.coli K12 (K12 Δ-ClpB) or Hafnia alvei AF036 (H. alvei), all inMueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl).

FIG. 19: Fat and lean mass gain from D0 in high fat diet (HFD)-inducedobese mice after 14 days of intragastric gavage with E. coli K12 (K12Δ-ClpB) or Hafnia alvei AF036 (H. alvei), all in Mueller-Hilton (MH)medium, or with MH medium only, as a control (Ctrl). A. Fat mass gain(in g). B. Lean mass gain (in g).

FIG. 20: Body weight gain (in AUC—Area Under the Curve) in obese fa/famice after 21 days of intragastric gavage with E. coli Niessle 1917(Nissle), E. coli K12 (K12 ΔClpB) or Hafnia alvei AF036 (H. alvei), allin Mueller-Hilton (MH) medium, or with MH medium only, as a control(Ctrl).

FIG. 21: Cumulative food intake (in AUC) in obese fa/fa mice after 21days of intragastric gavage with E. coli Niessle 1917 (Nissle), E. coliK12 (K12 ΔClpB) or Hafnia alvei AF036 (H. alvei), all in Mueller-Hilton(MH) medium, or with MH medium only, as a control (Ctrl).

FIG. 22: Fat mass gain (in g) in obese fa/fa mice after 21 days ofintragastric gavage with E. coli Niessle 1917 (Nissle), E. coli K12 (K12ΔClpB) or Hafnia alvei AF036 (H. alvei), all in Mueller-Hilton (MH)medium, or with MH medium only, as a control (Ctrl).

EXAMPLES Example 1 Material & Methods

In vitro E. coli Growth After Regular Nutrient Supply

The E. coli K12 bacteria were cultured at 37° C. in 40 mL of MH medium(Becton, Dickinson, Md.) containing 30% beef infusion, L75% caseinhydrolysate and 0.15% starch with pH 7.3 at 25° C. in 50 mL Falconvials. For modeling two scheduled daily meals in humans, bacteriareceived new MH medium every 12 h during 5 consecutive days. Bacterialgrowth was measured as an OD at λ=600 nm by a spectrophotometer every 2h after the 1^(st) provision of MH medium, every 1 h after the 3^(rd)and every 10 min after the 5^(th) provisions. At the end of each 12 hcycle, bacteria were centrifuged for 5 min at 6,000 rpm at roomtemperature (RT). The supernatants were discarded and replaced by anequivalent volume (˜40 mL) of a new MH medium. After the lastsupplementation of MH medium, bacteria were sampled for proteinextraction in the exponential phase and in the following stationaryphase.

Protein Extraction

E. coli K12 bacteria were centrifuged at 4° C. for 30 min at 4,000 g.Bacterial residues were dissolved in 2 mL oftrishydroxymethylaminomethane (TRIS) buffer (pH 7.4) and homogenized bysonication for 3 min at RT. To separate proteins from the undissolvedcell fragments, the bacterial homogenate was centrifuged at 4° C. for 30min at 10,000 g. The supernatant was recovered and then ultracentrifugedat 4° C. for 45 min at 60,000 g to further separate proteins intocytoplasmic (supernatant) and membrane (residues) fractions. Membraneproteins were dissolved in TRIS buffer (pH 7.4). Protein concentrationswere measured using 2-D Quant Kit (GE Healthcare, Piscataway, N.J.).

Two-Dimensional Polyacrylamide Gel Electrophoresis

For 2D-PAGE, 300 μg of E. coli protein extract were used to rehydrateimmobilized pH gradient (IPG) strips (pH 4-7; 18 cm; BIO-RAD, Hercules,Calif.). Proteins were then resolved in the first dimension byisoelectric focusing for a total of 85,000 V-h by using the IPGphorisoelectric focusing system (GE Healthcare). After focusing, IPG stripswere incubated for 15 min in the equilibration buffer [urea 6 mol/L, 30%(v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), Tris-HCl 50mmol/L pH 8.8, and 0.25% (w/v) bromophenol blue containing 2% (w/v)dithiothreitol] and then alkylated for 15 min in the equilibrationbuffer containing 4% (w/v) iodoacetamide. IPG strips were subsequentlyaffixed onto 10% polyacrylamide gradient gels (20 cm 18 cm 1 mm) forSDS-PAGE. The second dimension was performed overnight in the EttanDaltsix vertical electrophoresis system (GE Healthcare) with 12 mA/gelat 25° C. After SDS-PAGE, the 2D gels were fixed for 2 h in 2% (v/v)orthophosphoric acid and in 50% (v/v) methanol at RT. Gels were thenrinsed with water, and the protein spots were visualized by CBB G-250(BIO-RAD) staining [34% (v/v) methanol, 17% (w/v) ammonium sulfate, 2%(v/v) orthophosphoric acid, and 0.66 g CBB G-250/L].

Analysis of Differential Protein Expression

Images of stained 2D gels were scanned by an ImageScanner II (GEHealthcare) calibrated with a grey scale marker (Kodak, Rochester, N.Y.)and digitalized with Labscan 6.0 software (GE Healthcare). Analysis ofdifferential protein expression including spot detection,quantification, matching, and comparative analysis was performed usingImageMaster 2D Platinum 5.0 software (GE Healthcare). Each proteinsample was subjected to 2D-PAGE at least 3 times (membrane proteins) and4 times (cytoplasmic proteins) to minimize run-to-run variation, andeach set of 3 (or 4) gels was compared using ImageMaster to confirm thenonappearance of statistically differential spots within the set ofgels. The most representative gel (gel migration, spot definition, andspot number) of each set was used to compare E. coli proteins betweenexponential and stationary phases. The expression level was determinedby the relative volume of each spot in the gel and expressed as %volume, calculated as spot volume/Σvolumes of all spots resolved in thegel. This normalized spot volume takes into account variations due toprotein loading and staining by considering the total volume over allthe spots present in the gel. Variations in abundance were calculated asthe ratio of average values of % volume for a group of spots between the2 phases. Only spots with a volume variation ratio >1.5 were consideredrelevant. The absence of a spot within a gel indicated that nodetectable expression could be reported for a protein under the selectedexperimental condition. The corresponding p values were determined byStudent's t-test (significance level p<0.05) after spot volumelog-transformation.

Protein Identification by Liquid Chromatography-Electrospray IonizationMS/MS

The protein spots of interest were excised from CBB G-250-stained 2Dgels using the Ettan Spot Picker (GE Healthcare), and automated in-geldigestion of proteins was performed on the Ettan Digester (GEHealthcare) as previously described (Goichon et al., 2011). Proteinextracts were then resuspended in 10 μL of 5% (v/v) acetonitrile/0.1%(v/v) formic acid and then analyzed with a nano-LC1200 system coupled toa 6340 Ion Trap mass spectrometer equipped with a nanospray source andan HPLC-chip cube interface (Agilent Technologies, Courtaboeuf, France).Briefly, peptides were enriched and desalted on a 40 nL RP-C18 trapcolumn and separated on a Zorbax (30-nm pore size, 5-μm particle size)C18 column (43 mm long x 75 μm inner diameter; Agilent Technologies). A9-min linear gradient (3%-80% acetonitrile in 0.1% formic acid) at aflow rate of 400 nL/min was used and the eluent was analyzed with an iontrap mass spectrometer.

For protein identification, MS/MS peak lists were extracted and comparedwith the protein databases by using the MASCOT Daemon version 2.2.2(Matrix Science) search engine. The searches were performed with thefollowing specific parameters: enzyme specificity, trypsin; one missedcleavage permitted; no fixed modifications; variable modifications,methionine oxidation, cysteine carbamidomethylation, serine, tyrosineand threonine phosphorylation; monoisotopic; peptide charge, 2+ and 3+;mass tolerance for precursor ions, 1.5 Da; mass tolerance for fragmentions, 0.6 Da; ESI-TRAP as instrument; taxonomy, E. coli; National Centerfor Biotechnology Information (NCBI) database [NCBInr 20120531 (18280215sequences, 6265275233 residues)] (Bethesda, Md.). Protein hits wereautomatically validated if they satisfied one of the following criteria:identification with at least two top ranking peptides (bold and red)each with a MASCOT score of more than 54 (p<0.01), or at least two topranking peptides (bold and red) each with a MASCOT score of more than 47(p<0.05). To evaluate false-positive rates, all the initial databasesearches were performed using the “decoy” option of MASCOT. Results wereconsidered relevant if the false-positive rate never exceeded 1%.

ATP Assay

In vitro ATP production was measured using ATP colorimetric/fluorometricassay kit according to the manufacturer's instructions (BioVision, CA).Briefly, bacterial proteins from the exponential or stationary phaseswere placed into a series of wells in duplicates for each concentration(1, 10 and 25 μg/mL in ATP assay buffer) and adjusted to 50 μL/well withATP assay buffer. Then 10 μL of different nutrients solution, 15%sucrose or MH medium were added to corresponding wells and adjusted to50 μL/well with the ATP assay buffer; 50 μL/well of ATP buffer only wasadded to control wells. The plate was incubated 2 h at 37° C. After theincubation, 50 μL of ATP reaction mix (containing ATP assay buffer, ATPprobes, ATP converter and developer mix) were added in each well. OD wasmeasured at 570 nm after 30 min of incubation at RT, protected from daylight.

Development and Validation of ClpB Immunoassay

Design of the ClpB detection assay was based on several criteria, suchas a specific and sensitive detection in a linear concentration range. Aparticular condition was the ClpB detection without α-MSHcross-reactivity, which may occur due the presence of α-MSH-likeepitope(s) in the ClpB molecule. For simplicity of the procedure andsignal detection, we used a standard 96-well ELISA plate and an optionof the OD reading by a spectrophotometer. The detailed protocols of theClpB ELISA and Western blot (WB) are presented in separate sections.

To prevent binding of the Revelation antibody (Ab) to the Capture Ab, weproduced ClpB Capture Ab and ClpB Detection Ab in different species,rabbit and mouse, respectively. For the most efficient capture of theClpB protein from complex biological samples, we coated the ELISA platewith rabbit polyclonal Ab having multiple anti-ClpB epitopes. To avoidcross-reactivity between ClpB and α-MSH we used, as the Detection Ab, amouse monoclonal anti-ClpB Ab that has been characterized by highsensitivity and specificity to recognize ClpB but not α-MSH pre-selectedby ELISA screening of several Ab clones.

Alkaline phosphatase-conjugated anti-mouse Revelation Ab was used as acommon ELISA tool to obtain a chromogenic enzymatic reaction readable asan OD proportional to the analyte concentration. A linear change in ODresulting from the detection of 7 consecutive dilutions of a recombinantE. coli ClpB protein ranging from 2 pM to 150 pM was obtained withoutreaching a plateau and without saturation of the OD signal.

To validate the specificity of the developed ClpB assay, we measuredClpB concentrations in protein samples extracted from 10 differentcultures of E. coli K12 WT and from a culture of ΔClpB mutant E. colibacteria. ΔClpB mutant and the corresponding wild type (WT) strains werekindly provided by Dr. Axel Mogk (ZMBH, Heidelberg University, Germany).Furthermore, we analyzed the ClpB presence in these bacterial proteinsamples by WB using anti-ClpB polyclonal rabbit Ab and compared thesignal intensity values between the WB bands and ClpB concentrations inELISA. ClpB was detected in all WT E. coli cultures, with 7 cultureshaving ClpB concentrations higher than 1000 pM, while ClpB was notdetectable in the protein sample extracted from ΔClpB E. coli. WBrevealed a major band of an expected 96 KDa size in WT but not in ΔClpBE. coli. The OD intensity of these bands varied between individualsamples and correlated positively with ClpB concentrations measured byELISA in the same samples of E. coli cultures. Thus, the absence of ClpBdetection in protein preparations of ΔClpB E. coli confirmed thespecificity of the assays, and a good accordance between the ELISA andWB provided the cross-validation of both ClpB immunodetectiontechniques.

To verify that ClpB plasma assay detects ClpB derived from gut bacteria,we used the ClpB ELISA to measure CipB in plasma of mice which had beensupplemented via intragastric gavage daily for 3 weeks with WT or withΔClpB E. coli. Plasma samples were available from our previouslypublished study (Tennoune et al., 2014). We found that ClpB was normallypresent in mouse plasma including both controls and mice gavaged with aculture broth without bacteria. Importantly, ClpB plasma levels wereincreased in mice receiving WT E. coli but were unchanged in micesupplemented with ClpB-deficient E. coli, confirming the gut bacterialorigin of plasmatic ClpB.

ClpB ELISA

Rabbit polyclonal anti-E. coli ClpB antibodies (customly produced byDelphi Genetics, Gosselies, Belgium), were coated onto 96-well Maxisorpplates (Nunc, Rochester, N.Y.) using 100 μL and a concentration of 2μg/ml in 100 mM NaHCO3 buffer, pH 9.6 for 12 h at 4° C. Plates werewashed (5 min×3) in phosphate-buffered saline (PBS) with 0.05% Tween 20,pH 7.4. The recombinant E. coli ClpB protein (customly produced byDelphi Genetics), was diluted serially to 5, 10, 25, 50, 70, 100 and 150pM in the sample buffer (PBS, sodium azide 0.02%, pH 7.4) and added tothe wells in duplicates to make a standard. The analyte samplesincluded: colonic mucosa and plasma samples from mice and rats orproteins extracted from E. coli K12 cultures. Analyte samples were addedto the remaining wells in duplicates and were incubated 2 h at RT.Plates were washed (5 min×3) in PBS with 0.05% Tween 20, pH 7.4. Mousemonoclonal anti-E. coli ClpB antibodies (1:500 in sample buffer,customly produced by Delphi Genetics) were added to the wells andincubated 90 min at RT. Plates were washed (5 min×3) in PBS with 0.05%Tween 20, pH 7.4. Goat anti-mouse IgG conjugated with alkalinephosphatase (1:2000 in sample buffer) from Jackson ImmunoResearchLaboratories, Inc. (West Grove, Pa.) were added to the wells andincubated for 90 min at RT. Plates were washed (5 min×3) in PBS with0.05% Tween 20, pH 7.4 and then 100 μL of p-nitrophenyl phosphatesolution (Sigma, St. Louis, Mo.) was added as alkaline phosphatasesubstrate. After 40 min of incubation at RT, the reaction was stopped byadding 50 μL of 3N NaOH. The OD was determined at 405 nm using amicroplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). BlankOD values resulting from the reading of plates without addition ofplasma samples or ClpB protein standard dilutions were subtracted fromthe sample OD values.

CIpB Western Blot

Western blot was performed using proteins extracted from E. coli K12.Protein samples (10 μg) were separated on 20% acrylamide SDS gel inTris-Glycine buffer and transferred to a nitrocellulose membrane (GEHealthcare, Orsay, France), which was blocked for at least 1 h at RTwith 5% (w/v) non-fat dry milk in TBS (10 mmol/L Tris, pH 8; 150 mmol/LNaCl) plus 0.05% (w/v) Tween 20. Then, the membrane was incubatedovernight at 4° C. with rabbit polyclonal anti-E. coli ClpB antibodies(1:2000, Delphi Genetics). After three washes in a blocking solution of5% (w/v) non-fat dry milk in TBS/0.05% Tween 20, membranes wereincubated for 1 h with peroxidase-conjugated anti-rabbit IgG (1:3000,SantaCruz Biotechnology). After three washes, the peroxidase reactionwas revealed using the ECL detection kit (GE Healthcare). Protein bandswere compared with the molecular weight standard (Precision Plus,BioRad) and films were scanned using ImageScanner III (GE Healthcare)and analyzed for the band pixel density using the ImageQuant TL software7.0 (GE Healthcare).

Intestinal Administrations of E. coli Proteins in Rats

Animals

Animal care and experimentation were in accordance with guidelinesestablished by the National Institutes of Health, USA and complied withboth French and European Community regulations (Official Journal of theEuropean Community L 358, 18/12/1986). Female Sprague-Dawley rats, bodyweight 200-250 g (Janvier, Genest-Saint-Isle, France) were kept inholding cages (3 rats per cage) in a fully equipped animal facilityunder regulated environmental conditions (22±1° C., on a 12 h light-darkcycle with lights on at 7:30 a.m.) for 1 week in order to acclimatizethem to the housing conditions. Standard pelleted rodent chow (RM1 diet,SDS, UK) and drinking water were available ad libitum.

Experiment #1

This experiment was designed to evaluate the relevance of our in vitromodel of E. coli growth to in vivo situations of bacterial growth in thegut. Rats were anaesthetized by ketamine (75 mg/kg, Virbac, Carros,France)/xylazine (5 mg/kg, Bayer, Leverkusen, France) solution, 3:1vol., 0.1 mL/100 g body weight I.P. After laparotomy, colon wasmobilized by placing 2 ligatures: 1^(st) at the caecocolonic junctionand the 2^(nd), 4 cm below. Colonic infusions and luminal contentsampling were performed using a polypropylene catheter inserted into theascending colon and fixed with the 1^(st) ligature. 2 mL of MH medium orwater were gently infused into the colon and immediately thereafterwithdrawn for the measurement of OD. After OD measurement, the wholesample of the colonic content was returned into the colon. Such samplingof the colonic content, without adding new MH medium or water, wasrepeated every 5 min during first 20 min and then at 30 min and 60 min.Bacterial density was measured as an OD at λ=600 nm by aspectrophotometer. Blood samples were taken from the portal vein beforeand 30 and 60 min after the 1^(st) infusion. Fecal samples were takenfrom the colon at the end of experiment for DNA extraction and PCR ofClpB.

Real-Time Quantitative Polymerase Chain Reaction

Quantitative PCR (qPCR) was performed to analyze bacterial density ofClpB DNA expressing bacteria using a CFX 96 q-PCR instrument (BioRad,CA). Total DNA was extracted from the rat feces using QAMP DNA stoolmini kit (QIAGEN Venlo, Netherlands). The qPCR mix included 5 μL of SYBRGreen Master (QlAgen, West Sussex, UK), 0.5 μM each of forward andreverse primers, DNA from samples and water to give a total volume of 10μL. The primers were purchased from Invitrogen (Cergy-Pontoise, France).A three-step PCR was performed for 40 cycles. The samples were denaturedat 95° C. for 10 min, annealed at 60° C. for 2 min, and extended at 95°C. for 15 s.

Experiment #2

This experiment aimed to evaluate effects of E. coli proteins onintestinal peptides (GLP-1 and PYY) release in the systemic circulation.Rats were anaesthetized and the colon was mobilized as described above,colonic infusions of E. coli proteins (0.1 μg/kg of protein in 2 mL ofPBS) extracted in the exponential (n=6) or in the stationary phase (n=6)were preformed once for 20 min. Blood samples were taken from the portalvein before and after 20 min of colonic infusions for assays of GLP-1,PYY and ClpB. Samples of colonic mucosa were taken at the end ofexperiment for ClpB assay. GLP-1 and PYY assays were performed using afluorescent enzyme immunoassay kit (Phoenix Pharmaceutical Inc., CA),according to the manufacturer's instructions. The fluorescence wasmeasured at 325 nm for excitation and 420 nm for emission using amicroplate reader Chameleon (HIDEX Inc., Turku, Finland).

Administrations of E. coli Proteins in Rats, Food Intake and Brain c-fosStudy

Animals

Male Wistar rats, body weight 200-250 g (Janvier, Genest-Saint-Isle,France) were acclimatized to the housing conditions and were fed asdescribed above. Three days before experiments, the rats weretransferred to individual metabolism cages (Tecniplast,

Lyon France) where they were fed ad libitum with the same RM1 diet butin powdered form (SDS). Drinking water was always available. The ratswere gently handled daily for several min during the acclimation periodto habituate them to manipulations. At the end of acclimation, rats weredistributed into three groups to achieve similar mean body weight andwere used in the Experiments 1-3. Two experiments including foodrestriction were performed in the same rats with 4 days' interval. The3^(rd) experiment in free feeding rats involved their new series.

Experiment #1

The 1^(st) experiment was aimed to compare effects of membrane proteinsof E. coli extracted in exponential and stationary phases. Rats weredeprived from food overnight (between 18.00 h and 10.00 h), while waterwas available ad libitum. On following day after food deprivation, E.coli proteins were injected I.P. at 10.00 h and rats immediatelyreturned to their metabolism cages, which contained a pre-weighed amountof food. Food intake was measured at 1, 2, and 4 h. The 1^(st) group ofrats (n=6) received 0.1 mg/kg of membrane proteins extracted from E.coli in exponential phase in 300 μL of PBS; the 2^(nd) group of rats(n=6) received 0.1 mg/kg of membrane proteins extracted from E. coli instationary phase and the control group (n=6) received 300 μL of PBS.

Experiment #2

The 2^(nd) experiment was aimed to compare effects of cytoplasmicproteins of E. coli extracted in exponential and stationary phases. Asimilar experimental protocol was used as that for Experiment #1.

Experiment #3

This experiment was designed to evaluate effects of total E. coliproteins on food intake in free feeding rats. Injections of E. coliproteins (0.1 mg/kg of protein in 300 μL PBS, I.P.) extracted in theexponential (n=6) or in the stationary phase (n=6) or PBS only ascontrol (n=6), were carried out at 19.30 h and the animals were returnedto their metabolism cages which contained a pre-weighed amount of food.Cumulative food intake was measured after 2 h. Immediately thereafter,rats were anaesthetized by sodium pentobarbital (0.2 mg/kg, I.P.) andperfused for the immunohistochemical study of c-fos expression in thebrain.

Tissue Preparation and Immunohistochemistry

Brains were fixed by perfusion/immersion in 4% paraformaldehyde, frozenand cut (14 μm) on a cryostat (Leica Microsystems, Nanterre, France) andthen processed for immunohistochemistry using a tyramide signalamplification (TSA) plus fluorescein kit (NEN, Boston, Mass.). Forsingle staining, rabbit polyclonal antisera against c-fos (1:4,000,Ab-5, Calbiochem, Merck KGaA, Darmstadt, Germany) was used. For doublestaining, following the TSA, a direct immunofluorescence technique wasapplied using either rabbit monoclonal antibodies against β-endorphin(β-end) 1:1,000 (Life Technologies, Frederick, Md.) which was revealedby anti-rabbit Cyanine-3 antibodies 1:200 (Jackson ImmunoResearch, WestGrove, Pa.) or mouse monoclonal IgG against calcitonin gene-relatedpeptide (CGRP) 1:1,000 (Santa Cruz Biotechnology, Inc., TX) which wasrevealed by anti-mouse rhodamine red-conjugated antibodies 1:200(Jackson ImmunoResearch). In the hypothalamic arcuate and ventromedialnuclei and in the central nucleus of amygdala, positive cells werecounted at ×20 magnification from six consecutive sections. The meannumber of positive cells per rat was used to calculate the group mean.Digital images were optimized for brightness and contrast in AdobePhotoshop 6.0 software (Adobe Systems, San Jose, Calif.).

Chronic Administrations of E. coli Proteins in Mice

Two-month-old male C57B16 mice (n=32) were purchased from Janvier Labsand acclimated to the animal facility for 1 week with 12 h light-darkcycle, lights on at 8:00. Then, the mice were placed individually in theBioDAQ mouse cages (Research Diets, Inc., New Brunswick, N.J.), eachequipped with an automatic feeding monitor. After 3 days of acclimationto the BioDAQ cages, mice were divided into three groups (n=8), eachreceiving different treatments consisting of two daily I.P. injectionsat 9:00 and at 18:30 of either: (i) PBS, (ii) bacterial proteinsextracted in exponential phase (0.1 mg/kg of body weight), (iii)bacterial proteins extracted in stationary phase (0.1 mg/kg of bodyweight). Food (SERLAB, Montataire, France) and drinking water wereavailable ad libitum and body weight was measured daily. Feeding datawere continuously monitored for one week and analyzed using the BioDAQdata viewer 2.3.07 (Research Diets). For the meal pattern analysis, theinter-meal interval was set at 300 s. The satiety ratios were calculatedas time (s) of post-meal interval divided by amount of food (g) consumedin the preceding meal. After the experiment, mice were killed bydecapitation; the brain was removed and the hypothalamus dissected forthe neuropeptide mRNA microarray.

Hypothalamic Neuropeptide mRNA Microarray

Total RNA was extracted from the hypothalamus of mice, which receivedchronic administrations of E. coli proteins, using the NucleoSpin® RNAII kit (Macherey-Nagel, Duren, Germany) following the manufacturers'protocol. Digested RNA were reverse-transcribed at 42° C. for 60 minusing the ImProm-II™ Reverse Transcription System kit (Promega, Madison,Wis.). Obtained cDNA was used in real-time PCR reaction. A panel of 9primer pairs was designed with the Primer express software (LifeTechnologies,

Saint-Aubin, France) and validated for efficiency and specificity. ThePCR reaction, composed of 6 μL of cDNA and 6 μL of Fast SYBR GreenMaster Mix (Life Technologies) containing specific reverse and forwardprimers at a concentration of 100 nM, was dispensed in 96-well plateswith a Bravo liquid handling system (Agilent) and amplified with aQuantStudio 12K Flex (Life Technologies). The cDNA-generated signals fortarget genes were internally corrected with that of reference genesignals for variations in amounts of input mRNA. Gene expression levelwas then compared to a corresponding control sample group, andregulations were determined with the 2^(−ΔΔCq) method.

Electrophysiological Recordings

Brain slice (250 μm) were prepared from adult POMC-eGFP mice (5-7 weeksold; Ref: C57BL/6J-Tg(Pomc-EGFP)1Low/J, The Jackson Laboratory) aspreviously described (Fioramonti et al., 2007). Slices were incubated atRT, in oxygenated extracellular medium containing (in mM): 118 NaCl, 3KCl, 1 MgCl₂, 25 NaHCO3, 1.2 NaH₂PO₄, 1.5 CaCl2, 5 HEPES, 2.5 D-glucose(osmolarity adjusted to 310 mOsM with sucrose, pH 7.4) for a recoveryperiod at least 60 min. Once in the recording chamber, slices wereperfused at 2-3 mL/min with the same extracellular medium. Slices wereviewed with a Nikon microscope EF600 (Nikon France, Champigny sur Marne)outfitted for fluorescence (fluorescein filter) and IR-DIC videomicroscopy. Viable ARC POMC neurons were visualized using a ×40 waterimmersion objective (Nikon) with a fluorescence video camera (Nikon).Borosilicate pipettes (4-6 MΩ; 1.5 mm OD, Sutter Instruments, Novato,Calif.) were filled with filtered extracellular medium. Cell-attachedrecordings were made using a Multiclamp 700B amplifier, digitized usingthe Digidata 1440A interface and acquired at 3 kHz using pClamp 10.3software (Axon Instruments, Molecular Devices, Sunnyvale, Calif.).Pipettes and cell capacitances were fully compensated. After a stablebaseline was established, 1 nM of ClpB (Delphi Genetics) was perfusedfor 5-10 min. The POMC neurons' firing rate was measured over the last 3min of the ClpB perfusion, 7-10 min after the perfusion and comparedwith the firing rate measured 3 min before the perfusion.

Statistical Analysis

Data were analyzed and the graphs were plotted using the GraphPad Prism5.02 (GraphPad Software Inc., San Diego, Calif.). Normality wasevaluated by the Kolmogorov-Smimov test. Group differences were analyzedby the analysis of variance (ANOVA) or the non-parametric Kruskal-Wallis(K-W) test with the Tukey's or Dunn's post-tests, according to thenormality results. Where appropriate, individual groups were comparedusing the Student's t-test and Pearson's correlations or theMann-Whitney (M-W) test according to the normality results. Effects ofcontinuous experiments were analyzed using repeated measurements (RM)ANOVA and the Bonferroni post-tests. Data shown as means±standard errorof means (s.e.m), and for all test, p<0.05 was considered statisticallysignificant.

Results

-   E. coli Growth after Regular Nutrient Provision

After the 1^(st) provision of the Miieller-Hinton (MH) nutritionalmedium to E. coli culture, three phases of bacterial growth wereobserved: 1) lag phase of 2 h; 2) exponential growth phase of 4 h and 3)stationary phase with the 0.35 optical density (OD) remaining stable for6h. After the 3^(rd) and 5^(th) MH medium supplies, only two growthphases were found: the exponential and stationary, the 1^(st) phasestarting immediately after the nutrient provision. Each new feedingcycle was characterized by a shorter duration of the exponential growthphase: 2 h after the 3^(rd) and 20 min after the 5^(th) provision. Afterthe 5^(th) nutrient provision, bacterial proteins were extracted andseparated into the membrane and cytoplasmic fractions used for theproteomic analysis and in vivo experiments in fasted rats. In the newexperiment, to verify if continuing regular nutrient provision mayfurther accelerate the dynamics of bacterial growth, E. coli K12 weresupplied with nutrients 9 times. We found that, after the 7^(th) and9^(th) nutrient provisions, the exponential growth phase did not furtherchange, lasting for 20 min with the same (Δ0.3) relative increase in OD,reflecting an identical bacterial growth after each supply of nutrients.According to the McFarland standards, an increase of 0.3 OD correspondsto an increment of 10⁸-10⁹ of bacteria. After the 9^(th) nutrientprovision, bacterial proteins were extracted in the exponential andstationary phases, showing total protein concentrations of 0.088 mg/mLand 0.15 mg/mL, respectively. The extracted proteins were tested forClpB levels and have been used in the ATP production assay and for invivo experiments including intracolonic infusions and systemicinjections in free feeding mice and rats followed by the c-fos detectionin the brain.

Proteomic Analysis

To analyze whether the protein expression profiles vary according tonutrient-induced bacterial growth phases, two-dimensional polyacrylamidegel electrophoresis (2D-PAGE) was performed separately on membrane andcytoplasmic fractions of E. coli K12 proteins extracted 10 min and 2.3 hafter the 5^(th) addition of MH medium, corresponding to the exponentialand stationary phases, respectively. The total number of detectedprotein spots was 2895 (1367 membrane and 1528 cytoplasmic).

Comparison of the 2D-PAGE of membrane proteins between the exponentialand stationary phases revealed 20 differentially (by at least 1.5-fold)expressed proteins. Among them, 17 proteins showed increased expressionin the exponential phase and 15 were identified by mass spectrometry.Comparison of 2D-PAGE of cytoplasmic proteins showed 20 differentially(by at least 1.5-fold) expressed proteins. Contrary to the membraneproteins, the majority (19) of cytoplasmic proteins showed increasedexpression during the stationary phase. Only one protein spot,corresponding to flagellin, had higher expression in the exponentialphase. The majority of identified proteins were implicated in eitheranabolic or catabolic processes showing an overall mixt metabolicprofile in both growth phases.

ATP Production by E. coli Proteins In Vitro

To study if bacterial proteome change during growth phases may influencetheir energy extraction capacities, the ATP production from nutrients byE. coli K12 proteins of the exponential and stationary phases was testedin vitro. We found that proteins from both growth phases were able toincrease ATP production from different energy sources. The ATPconcentrations were higher when a protein-containing mixed energysource, such as the MH medium, was used as compared to a sucrosesolution. The ATP production increased dose-dependently withconcentrations of bacterial proteins, however, no significantdifferences were found between ATP-producing effects of proteins fromthe exponential or stationary phases.

ClpB Production by E. coli In Vitro

We developed and validated an enzyme-linked immunosorbent assay (ELISA)for detection of E. coli ClpB, which has been used in this study.Whether ClpB protein production is different between bacterial growthphases was studied in 4 separate E. coli K12 cultures. The Western blotdetected ClpB-corresponding 96 kDa bands in all proteins preparationswith increased levels during the stationary phase. These changes havebeen further confirmed using the ClpB ELISA in the same bacterialprotein preparations, showing that ClpB concentrations almost doubled inthe stationary phase.

Intestinal Infusions of Nutrients and E. coli Proteins

To verify if our in vitro model of nutrient-induced E. coli growth isrelevant to gut bacterial growth dynamics in vivo, MH medium or waterwere infused into the colon of anaesthetized rats. We found thatinstillations of MH medium, but not water, induced bacterialproliferation in the gut with the exponential growth phase lasting for20 min, consistent with the in vitro data. Plasma ClpB levels measuredin the portal vein were not significantly different 30 or 60 min afterMH infusion. Nevertheless, plasma ClpB concentrations correlatedpositively with ClpB DNA content in feces.

Next, to determine if growth-dependent changes of E. coli proteomes mayinfluence host mechanisms of appetite control locally in the gut, in aseparate experiment, anaesthetized rats received 20 min colonicinfusions of E. coli proteins from the exponential or stationary phases,both at 0.1 mg/kg. The concentrations of ClpB in the colonic mucosameasured 20 min after the infusion were higher in rats receiving thestationary phase proteins, however, plasma levels of ClpB were notaffected by bacterial proteins from either exponential or stationaryphases. Consistent with our hypothesis that effects of E. coli proteinson host appetite signaling might depend on the bacterial growth phase,we found that colonic instillations of E. coli proteins from theexponential but not the stationary phase stimulated plasma levels ofGLP-1 and, in contrary, increased plasma levels of PYY were detectedafter infusion of proteins from the stationary but not the exponentialphase.

Food Intake and Brain c-fos after Acute E. coli Proteins Administrationsin Rats

Because ClpB was present in plasma in all tested rats and mice, it ispossible that plasmatic E. coli proteins might influence appetite viatheir systemic action and that such effects can be different forproteins associated with bacterial growth phases. By testing thispossibility in overnight fasted rats, we found that a singleintraperitoneal (I.P.) administration (0.1 mg/kg of body weight) of themembrane fraction of E. coli proteins extracted in the stationary phase,decreased 1 h- and 2 h-food intake during refeeding as compared with thecontrol group. In contrast, administration of the cytoplasmic fractionof E. coli proteins (0.1 mg/kg of body weight, I.P.) extracted in theexponential phase increased 4 h food intake during refeeding.

To further investigate if E. coli total proteins may influencespontaneous food intake in a growth phase-dependent way, and to activatecentral sites such as the ARC, free feeding rats were injected beforethe onset of the dark phase with bacterial proteins (0.1 mg/kg of bodyweight, I.P.). Food intake was measured for 2 h following injections,and then the rats were killed for the analysis of c-fos expression inthe brain. We found that rats injected with bacterial proteins from thestationary phase ate less than controls, while food intake was notsignificantly affected by injections of bacterial proteins from theexponential phase.

Two hours after I.P. injections of E. coli proteins to free feedingrats, c-fos expression was immunohistochemically analyzed in the ARC andthe ventromedial nucleus (VMN) of the hypothalamus and in the CeA. Theincreased number of c-fos-positive cells was found in the ARC and VMN ofmice receiving bacterial proteins from the stationary phase. Themajority of c-fos expressing cells in the ARC contained β-endorphin(Controls, 71.31±12.81%, E. coli exp. phase, 73.56±10.45%, E. coli stat.phase, 80.50±9.68%, ANOVA p=0.36), i.e. were identified as anorexigenicPOMC neurons. Accordingly, the percentage of remaining c-fos neurons inthe ARC did not significantly differ among the groups (Controls,28.69±12.81%, E. coli exp. phase, 26.44±10.45%, E. coli stat. phase,19.5+9.68%, ANOVA p=0.36). Although the total numbers ofβ-endorphin-positive cells were not significantly different among thegroups (Controls, 54.82±10.67 cells, E. coli exp. phase, 66.03±11.43cells, E. coli stat. phase, 66.03±5.06 cells, ANOVA p=0.09), therelative number of activated β-endorphin neurons was increased in ratsreceiving stationary phase proteins as compared to controls and to ratsreceiving exponential phase proteins. Furthermore, the number ofactivated β-endorphin neurons correlated inversely with food intake(Pearson's r=−0.57, p=0.018).

In the CeA, the number of c-fos-positive cells was increased in ratsinjected with proteins from the stationary phase, as compared to the twoother groups. Almost all c-fos-positive cells in the CeA were phenotypedas CGRP-expressing neurons (Controls, 100±0.01%, E. coli exp. phase,100±0.01%, E. coli stat. phase, 100±0.01%, ANOVA p=0.92). Although thetotal numbers of CGRP-positive neurons in the CeA were similar among thegroups (Controls, 123.8±13.15 cells, E. coli exp. phase, 118.3±25.59cells, E. coli stat. phase, 126.1±6.64 cells, ANOVA p=0.85), thepercentage of c-fos activated CGRP neurons was increased only in ratsreceiving stationary phase proteins. Activation of CGRP neuronscorrelated inversely with food intake (Pearson's r=−0.89, p=0.001).

Feeding Pattern and Hypothalamic Neuropeptides After Chronic E. coliProtein Injections in Mice

To determine if bacterial proteins may influence feeding pattern, twodaily injections of E. coli total proteins (0.1 mg/kg of body weight,I.P.) were administered for one week to free feeding mice. The first dayafter injections was characterized by significantly lower body weightand food intake in mice receiving bacterial proteins from the stationarybut not the exponential phase as compared to controls. Although dailymeal size was not significantly different among the groups, its decreaserelative to the day before injections was observed one week later inmice receiving stationary phase proteins. Meal frequency was notsignificantly different among the groups, although a trend towards anincrease was observed in mice receiving bacterial proteins from thestationary phase. Although total food intake among 3 groups was notsignificantly different during 6 days of injections, analyzing itseparately for the light and dark periods, showed that mice injectedwith the exponential phase proteins had increased food intake during thelight period, but it was decreased during the dark period. In contrast,mice receiving the stationary phase proteins displayed lower thancontrols food intake in the dark period without any effect in the lightperiod. During the first day after injections, the satiety ratio wasincreased in mice receiving proteins from the stationary phase, and thesame group showed a tendency towards a decrease, one week later.

To get an insight into the molecular changes underlying altered feedingpattern observed after 6 days of bacterial protein injections, weanalyzed hypothalamic mRNA expression levels of several neuropeptidesinvolved in appetite control. We found that mice receiving thestationary phase proteins showed elevated precursor mRNA levels ofbrain-derived neurotrophic factor (BDNF) and orexin as compared tocontrols, and of corticotropin-releasing hormone (CRH) as compared tomice injected with proteins in the exponential phase, which also showedelevated levels of BDNF but decreased QRFP.

Electrophysiological Activation of Hypothalamic POMC Neurons by ClpB

To determine whether ClpB, as a marker of E. coli proteins up-regulatedin the stationary growth phase and a mimetic of α-MSH, activates ARCPOMC neurons, ClpB effect was examined on brain slices from POMC-eGFPmice using cell-attached patch-clamp electrophysiology. Bath applicationof ClpB (1 nM) increased action potential frequency of ˜50% of ARC POMCneurons (n=7/13), by 229±109% (basal: 2.02±0.78 Hz vs. ClpB: 3.82±1.36Hz), as shown in FIG. 2. In general, POMC neurons did not fully returnto their basal firing rates until at least 10 min after ClpBapplication.

These results thus suggest a direct effect of the ClpB protein onsatiation and satiety.

Discussion

Our study reveals that bacterial proteins may physiologically link gutbacteria to the host control of appetite including its both short- andlong-term mechanisms associated with the nutrient-induced bacterialgrowth in the gut and their systemic effects, respectively. Thefollowing main results support this conclusion: 1) regular provision ofnutrients accelerates and stabilizes the exponential growth of E. colilasting for 20 min, in agreement with the in vivo data; 2) E. colistationary growth phase was characterized by increased total bacterialprotein content and a different proteome profile, including increasedClpB; 3) E. coli proteins from both growth phases dose-dependentlystimulated in vitro ATP production; 4) Plasma levels of ClpB did notchange after nutrient-induced bacterial growth in the gut, butcorrelated with ClpB DNA in gut microbiota; 5) Intestinal infusion of E.coli proteins from the exponential and stationary growth phasesstimulated plasma GLP-1 and PYY, respectively. 6) Systemic injections ofE. coli proteins decreased significantly food intake only by theproteins from the stationary phase, accompanied by c-fos activation inanorexigenic ARC and CeA neurons, and finally 7) ClpB stimulated firingrate of ARC POMC neurons.

Regular Provision of Nutrients and Bacterial Growth

Among the wide variety of bacteria that colonize the gastrointestinaltracts of humans, E. coli is the most abundant facultative anaerobe,justifying it as a model organism for commensal gut bacteria (Foucaultet al., 2009). Here, we found that E. coli change their dynamics ofgrowth during regular nutrient supply, resulting after the 5^(th)feeding cycle, in an immediate exponential growth lasting for 20 minfollowed by the stationary phase. The growth cycle is then identicallyreproduced after the next provision of nutrients, suggesting that it canplay a role of a pacemaker set by intrinsic to bacteria mechanisms. Asimilar dynamics of bacterial growth in response to nutrient infusionwas seen in the rat colon, supporting that our in vitro data can berelevant to in vivo situations, e.g. in humans taking regular meals. The10⁸-10⁹ increment of bacterial number remained stable after each newprovision, suggesting that the corresponding stable production of thebacterial biomass, including increased protein content in the gut, mayplay a role of a meal-induced regulatory factor for the host. Given thatthe average prandial phase in humans is similar to the duration of theexponential growth of regularly-fed bacteria, it is tempting tospeculate that the host satiety can be triggered by gut bacteriareaching the stationary phase, 20 min after a contact with ingestednutrients. However, bacterial content in the gastrointestinal tractranges from 10³ in the stomach to 10¹² in the colon. Moreover, about 2 his necessary for the advancement of ingested nutrients through thestomach and small intestine and about 10 h through the large intestine.Because of such a delay of nutrient delivery to most gut bacteria, it islikely that beside the direct contact with the nutrient bolus, bacterialgrowth during the prandial phase might be also initiated by nutrientsreleased into the gut lumen by the Pavlovian cephalic reflex toingestion.

Bacterial Protein Expression During Growth Phases and Intestinal Sensing

Because the growth dynamics of regularly-fed E. coli can be associatedwith the host prandial and postprandial phases, we have studied ifexpression of bacterial proteins may potentially link gut bacteria andhost control of appetite. First, we compared the proteomes of E. coli inthe exponential vs. stationary growth phases. For this analysis, E. coliproteins were extracted in the middle of the exponential phase, i.e. 10min after nutrient provision, and in the stationary phase 2 h later, atime normally characterized by feeling of satiety. The finding of atleast 40 differentially expressed proteins between two growth phasesconfirmed that they differ not only quantitatively, by protein content,which almost doubled after bacterial proliferation, but alsoqualitatively. The possible relevance of the modified protein expressionprofile to the host control of appetite has been then studied: i) bycomparing the ability of bacterial proteins to generate energysubstrate, and ii) by determining possible direct effects of bacterialproteins on appetite-regulatory pathways. The late possibility issupported by the recent proteomic identification of E. coli ClpB as aconformational protein mimetic of α-MSH (Tennoune et al., 2014); thedata that validated our earlier hypothesis that gut bacterial proteins,displaying epitopes homologous to anorexigenic or orexigenic peptides,may be responsible for production of cross-reactive autoantibodies(Fetissov et al., 2008). It is, hence, conceivable that a combination ofsuch bacterial mimetic proteins may influence appetite directly,depending on the protein expression profile associated with thebacterial growth phase. By analyzing E. coli cultures and the intestinalmucosa, we found increased ClpB levels associated with the stationarygrowth phase. Increased ClpB may, hence, contribute to the activation ofanorexigenic pathways after nutrient-induced E. coli proliferation andincreased bacterial protein production. An important question is wherethe bacterial proteins, such as ClpB, may act on the appetite-regulatorypathways.

Although bacterial proteins are present in the intestinal mucosa (Haangeet al., 2012), their passage across the gut barrier has not beenextensively studied (Lim and Rowley, 1985). In theory, after spontaneousand induced bacterial lyses in the gut (Rice and Bayles, 2008),bacterial components can pass across the mucosal epithelial barrier byabsorption in enterocytes and by paracellular diffusion, regulated bythe enteric nervous system (Neunlist et al., 2012). For instance,lipopolysaccharide (LPS), which is released upon lysis of gram-negativebacteria, is naturally present in plasma of healthy humans and mice withhigher basal levels after consuming high fat diets (Cani et al., 2007).LPS plasma levels are also increased postprandially (Harte et al.,2012), but there are no such data about bacterial proteins. Here we showthat plasma ClpB levels remain stable in rats following bacterialproliferation in the gut or after intestinal infusion of bacterialproteins. It indicates that plasmatic ClpB, and most likely otherbacterial proteins present in plasma, are not acutely influenced bynutrient-induced bacterial proliferation, and hence, they cannot beinvolved in the short-term satiety signaling to the brain.

Nevertheless, plasma ClpB concentrations correlated with ClpB DNA in gutmicrobiota, suggesting that the number of ClpB-producing bacteria, whichshould be at long-term relatively independent from its fluctuationsrelated to the nutrient-driven bacterial growth, can be the main factorresponsible for the long-term maintenance of plasma ClpB levels. Thisconclusion is further supported by our data, obtained for validation ofClpB ELISA, showing increased plasma ClpB concentrations in micechronically gavaged with E. coli but not in mice that receivedClpB-mutant E. coli. It is hence, possible that gut bacterial proteinspresent in plasma, including ClpB, may act systemically linking thecomposition of gut microbiota with the long-term control of energymetabolism.

Effects of E. coli Proteins on ATP Production In Vitro

Energy exchange through the food chains represents a universal linkbetween all organisms (Yun et al., 2006). ATP, derived from nutrientcatabolism in both bacteria and animals, serves as the main energysubstrate for anabolic processes. Animals can sense changes in ATP viathe activity of adenosine-5′-monophosphate-activated protein kinase(AMPK), resulting in increased food intake, when ATP levels are low andvice versa (Dzamko and Steinberg, 2009). Hence, in the present work, wecompared the ability of E. coli proteins in exponential and stationaryphases to generate ATP in vitro. Indeed, many of the identified proteinsdisplayed anabolic or catabolic properties. We found that E. coliproteins are able to stimulate ATP-production in vitro, suggesting thatthey may continue to catalyze ATP production after the bacterial lysisin the gut. Although no differences in ability to produce ATP was foundbetween the proteins from the exponential and stationary phases, thebacterial protein concentration-dependent ATP production indicates thatincreased bacterial protein content, after nutrient-induced bacterialproliferation, should result in higher ATP synthesis. The relevance ofthe efficiency of gut microbiota to harvest energy for the hostmetabolism has been previously established by comparing obese and leanhumans and mice (Turnbaugh et al., 2006). Our data further corroboratethese results by showing the ability of E. coli proteins to generateATP. It also suggests that meal-induced bacterial proliferation may leadto increased intestinal ATP, which should contribute to the luminalsensing of energy availability and gut relaxation (Glasgow et al.,1998).

Intestinal Effects of E. coli Proteins on Satiety Hormones

Next, we studied if E. coli proteins in the gut may stimulate thesystemic release of gut satiety hormones such as GLP-1 and PYY (Adrianet al., 1985; Batterham et al., 2002; Beglinger and Degen, 2006; Flintet al., 1998). In fact, both hormones are produced by the same ordistinct enteroendocrine L-cells present throughout the intestine andabundant in the colon (Eissele et al., 1992), and hence, L-cells aredirectly exposed to bacterial proteins. We found differential effects ofE. coli proteins on GLP-1 and PYY release, showing stimulation of GLP-1by the proteins from the exponential and of PYY from the stationarygrowth phases. These results point to some similarities betweennutrient-induced bacterial growth and the known dynamics of meal-inducedrelease of GLP-1 and PYY. In fact, as was shown in humans, an acute peakof plasma GLP-1 occurs 15 min after an intragastric infusion of a liquidmeal, while a longer-lasting elevated plasma PYY starts between 15 and30 min after a meal (Edwards et al., 1999; Gerspach et al., 2011).Longer release of GLP-1 was associated with fat intake (van der Klaauwet al., 2013). Thus, the growth dynamics of regularly-fed gut bacteriafits temporally into the dynamics of GLP-1 and PYY release, suggestingan inductive role of gut bacteria, and specifically of E. coli proteins,in meal-induced signaling of intestinal satiety. A differential effectof E. coli proteins from the exponential phase to stimulate GLP-1, maypossibly reflect an incretin role of GLP-1 in glycemic control (Edwardset al., 1999; Steinert et al., 2014). A recent demonstration offunctional MC4R expressed by L-cells (Panaro et al., 2014), provide abackground for their possible activation by α-MSH-mimetic bacterialproteins. An increased production of ClpB during the E. coli stationaryphase, as well as elevated ClpB levels in the intestinal mucosa,associated with increased plasma PYY levels, suggest a direct role ofClpB in activation of PYY-producing L-cells in the colon. From the otherhand, so far non-identified E. coli proteins possibly upregulated duringthe exponential growth phase, may preferentially stimulate GLP-1secretion.

Systemic Effects of E. coli Proteins on Food Intake andAppetite-Regulating Brain Pathways

We showed here that peripheral injections of E. coli proteins to hungryor free-feeding rats and free-feeding mice changed their food intakedepending on the growth phase of E. coli. Considering that plasma ClpBwas not affected by intestinal infusion of nutrients but was stable overshort time, the systemic action of bacterial proteins should beinterpreted as relevant to their long-term modulatory effects onappetite. Furthermore, because of a short duration of the exponentialphase, bacterial proteins upregulated during the long-lasting stationaryphase should dominate in plasma, and hence, their systemicadministrations can better represent the physiological situations. The0.1 mg/kg concentrations of E. coli proteins used in these experimentswere similar with the effective satietogenic doses of peptide hormonessuch as leptin or PYY after peripheral administrations in humans orrodents (Batterham et al., 2002; Halaas et al., 1995; Heymsfield et al.,1999).

An increase of food intake in hungry rats during refeeding in lightphase was observed after administration of cytoplasmic proteins from theexponential phase and a decrease by the membrane proteins from thestationary phase. This experiment confirmed that different proteinmixtures from the same bacteria are able to increase or decrease foodintake by their systemic action. However, in a more physiologicalsetting, by testing effect of total E. coli proteins in free feedingrats in the dark phase, only a decrease in food intake was observedwhich was induced by proteins from the stationary phase.

Results of repeated injections in free feeding mice further support arole of E. coli proteins to promote negative energy balance. Indeed,first day of bacterial protein injections was accompanied by decreasedfood intake and body weight, being significant in mice receivingstationary phase proteins and who were also characterized by increasedsatiety ratio. Although food intake in these mice was normalizedthereafter, a progressive decrease of meal size was accompanied byincreased meal frequency, most likely as a compensatory mechanism tomaintain food intake (Meguid et al., 1998). Additionally, differentialeffects of E. coli proteins were observed during the light and darkphases. Accordingly, the pattern of mRNA expression ofappetite-regulating neuropeptides in the hypothalamus showed a mixtresponse with activation of both anorexigenic and orexigenic pathways.Of note, both groups of mice receiving E. coli proteins showed similarincrease of BDNF mRNA, an anorexigenic pathway downstream to MC4R in theVMN (Xu et al., 2003). This pathway may underlie a decreased food intakeduring the dark phase observed in both groups, and which was furtheraccentuated in mice injected with the exponential phase proteins showinglower levels of orexigenic QRFP (Chartrel et al., 2003) and NPY (Herzog,2003). On the contrary, mice that received bacterial proteins from thestationary phase, displayed an enhanced anorexigenic profile withelevated levels of CRH mRNA, most likely implicating MC4R-expressing PVNneurons (Lu et al., 2003). These changes combined with increased mRNAprecursor expression of orexin A that stimulates meal frequency (Bairdet al., 2009), may contribute to decreased meal size and satiety ratio,respectively, in these mice after 6 days of injections.

Consistent with our hypothesis that bacterial proteins produced duringthe stationary phase may activate some key central anorexigenicpathways, we found in free feeding rats an increased c-fos expression inthe anorexigenic ARC POMC neurons as well as in the VMN, which has longbeen known as a satiety center and is interconnected with the ARC POMCneurons (Steenson et al., 2005). The obtained c-fos pattern resemblesthat of a satietogenic response during food ingestion (Johnstone et al.,2006) or induced by satiety hormones such as PYY or pancreaticpolypeptide (Batterham et al., 2002; Challis et al., 2004; Lin et al.,2009). A relatively small number (˜10%) of c-fos-activated POMC neuronssuggests that circulating E. coli proteins might have a modulatoryeffect on appetite and body weight acting via these hypothalamicpathways. Although it was not feasible to determine c-fos activation bythe NPY/AgRP neurons, their contribution to signaling by bacterialproteins cannot be excluded; these neurons also express MC3R and MC4R(Mounien et al., 2005). Moreover, a stronger than in the ARC POMCactivations of c-fos in the CeA CGRP neurons (˜40%) may signify aconvergent down-stream action from ARC POMC and NPY/AgRP neurons andpossibly from other appetite-regulating brain areas, which has not beenanalyzed here, such as the nucleus of the solitary tract.

Finally, to determine if activation of appetite-regulating brain sites,such as the ARC POMC neurons, by bacterial proteins may be caused bytheir local action, we studied if application of ClpB on these neuronsmay activate their electrical activity. Our results showed that about ahalf of studied neurons increased their action potential frequency,remaining activated for at least 10 min. The sustained effect of ClpB isconsistent with the effect of α-MSH on POMC neurons expressingfunctional MC3R and MC4R (Smith et al., 2007), suggesting that ClpB canbe a physiological activator of the hypothalamic POMC neurons, somewhatsimilar to satietogenic PYY and leptin (Batterham et al., 2002; Cowleyet al., 2001). However, we do not know if ClpB was able to activate POMCneurons directly or via a local network.

Taken together, these data support a role of systemically present E.coli proteins which expression is increased in the stationary growthphase, such as ClpB, in promoting negative energy balance via activationof brain anorexigenic pathways. It also suggests that changes ofmicrobiota composition resulting in low or high abundance of E. coli,and possibly of other bacteria from the family of Enterobacteriaceae,may influence the host energy balance in a positive or negative ways,respectively.

Example 2

This example demonstrates the effect of ClpB-expressing bacteria on foodintake.

One-month-old male C57B16 mice (Janvier Laboratories) were acclimated tothe animal facility for 1 week and maintained as described above. Micewere distributed into 3 groups as follows: (i) gavaged with 10⁸ E. coliK12 bacteria (expressing ClpB); (ii) gavaged with 10⁸ E. coli K12bacteria deficient for ClpB; (iii) and controls that did not receive anytreatments. The ClpB mutant strain was generated in the Bernd Bukau'sLaboratory (ZMBH, Heidelberg University, Heidelberg, Germany) and waskindly provided together with the corresponding wildtype (WT) E. colibacteria by Dr. Axel Mogk. Mice were placed individually into the BioDAQcages (Research Diets) and intragastrically gavaged daily before theonset of dark phase for 21 days with 0.5 mL of LB medium with thebacteria. The first days of gavage were accompanied by a decrease inbody weight and food intake in mice receiving WT E. coli, contrary tothe bacteria that do not express the ClpB protein (FIG. 1).

Example 3

This example demonstrates the effect of ClpB-expressing bacteria onobese ob/ob mice.

Genetically obese ob/ob mice were acclimated to the animal facility for1 week and maintained as described above. Mice were intragastricallygavaged with (i) 10⁸ E. coli K12 bacteria (expressing ClpB); (ii) 10⁸ E.coli K12 bacteria deficient for ClpB; both in Mueller-Hilton (MH) mediumor with (iii) MH medium only, as a control. The ClpB mutant strain wasgenerated in the Bernd Bukau's Laboratory (ZMBH, Heidelberg University,Heidelberg, Germany) and was kindly provided together with thecorresponding wildtype (WT) E. coli bacteria by Dr. Axel Mogk. Mice wereplaced individually into the BioDAQ cages (Research Diets) andintragastrically gavaged daily for 21 days as indicated.

The inventors showed that gavage with E. coli K12 WT bacteria induced a56% reduction in weight gain (FIGS. 3 and 4), a reduced fat mass/leanmass ratio (FIGS. 5 and 6) and a reduction of 20% of the total foodintake (FIGS. 7 and 8), which was not observed with E. coli K12 bacteriadeficient for ClpB.

Example 4

This example demonstrates the effect of other strains of bacteriaexpressing ClpB on obese ob/ob mice.

Genetically obese ob/ob mice were acclimated to the animal facility for1 week and maintained as described above. Mice were intragastricallygavaged with (i) 10⁸ E. coli K12 bacteria (expressing ClpB); (ii) 10⁸ E.coli Niessle 1917 bacteria (expressing ClpB) (iii) 10⁸ E. coli Niessle1917 bacteria (expressing ClpB) in lyophilized form; all inMueller-Hilton (MH) medium or with (iv) MH medium only, as a control.Mice were intragastrically gavaged daily for 14 days as indicated.

The inventors showed that gavage with any strain of E. coliClpB-expressing bacteria induced a reduction in weight gain (FIGS. 9 and10) and a reduction in fat content (FIG. 11).

Example 5

This example confirms the direct satietogenic action of ClpB.

The probable effect of ClpB on feeding behavior via hypothalamic MCRreceptors family was shown in an animal study on rats, which receivedintracerebroventricular (ICV) injections of ClpB.

Sprague-Dawley male rats 200-250 g (Janvier, L'Arbresle, France) weremaintained at 24° C. with a 12:12-h light-dark cycle (light period 7-19h) in a specialized air-conditioned animal facility. Rats were fed withstandard pelleted chow (RM1 diet, SDS, Essex, UK). Drinking water wasalways available ad libitum.

For the implantation of the stainless-steel cannulas (C311 GA, diameterexternal 0.9 mm, internal 0.58 mm, Plastics One, Roanoke, Va.), ratswere anaesthetized by an intraperitoneal injection of ketamine (75mg/kg)/xylazine (5 mg/kg) mixture (3:1 vol-0.1 mL/100 g body weight) andplaced into a New Standard Stereotaxic Instrument for Rats and Mice(Stoelting Europe, Dublin, Ireland). The cannulas were implanted underan operating microscope (Carl Zeiss, Jena, Germany) into thehypothalamic paraventricular nucleus (Bregma: ±2.8 mm, lateral: −0.4 mmfrom the midline, and ventral: 8.2 mm from the dura mater), with theincisor bar set at −3.3 mm. The cannulas were fixed to the skull usingdental cement supported by the anchor screws. After awaking, rats werekept individually in the metabolism cages (Techniplast, Lyon, France)for 1 week and fed ad libitum with the same standard rodent chow (RM1,SDS) with water always available. Physical conditions and body weightgain were daily monitored in the postoperative period to ensure goodrecovery.

Then, the rats were placed individually into the BioDAQ rat cages(Research Diets, Inc., New Brunswick, N.J.), each equipped with anautomatic feeding monitor. After 3 days of acclimation to the BioDAQcages, rats were food deprived during 12 h before injection and dividedinto 3 groups (n=3), each receiving different doses of a singleinjection of ClpB: 10 ng, 100 ng and 1 mg, diluted in 2 mL of sterileartificial cerebrospinal solution. Injections were performed 15 minbefore the onset of the dark phase and food provision. Food intake wasmeasured during the dark phase.

Animals showed a dose dependent decrease of food intake (see FIG. 12).

Example 6

This example demonstrates the effect of C1pB-expressing Hafnia alvei onobese ob/ob mice.

Genetically obese ob/ob mice were acclimated to the animal facility for1 week and maintained as described above. Mice were intragastricallygavaged with (i) 10⁸ Hafnia alvei AF036 bacteria (expressing C1pB); (ii)10⁸ E. coli K12 bacteria deficient for ClpB; (iii) 10⁸ E. coli Niessle1917 bacteria (expressing ClpB); all in Mueller-Hilton (MH) medium orwith (iv) MH medium only, as a control.

The inventors show that treatment with Hafnia alvei induced asignificant decrease in body weight gain as compared to obese controls(FIG. 13). Moreover, the decrease in body weight gain was associatedwith a decrease of cumulative food intake (FIG. 14), and with a decreaseof both fat and lean masses, without alteration of the lean/fat massratio (FIG. 15).

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase thatis capable of hydrolyzing triacylglycerols, diacylglycerols,monoacylglycerols, and cholesteryl esters, as well as other lipid andwater soluble substrates. In particular, activation of HSL induceslipolysis. Hormonal activation of HSL occurs via cyclic AMP dependentprotein kinase (PKA), which phosphorylates HSL. An increase of theexpression of the phosphorylated form of HSL protein (pHSL) means thusactivation of lipolysis. Expression of pHSL was measured by westernblot.

The inventors showed that gavage with E. coli Niessle 1917 and Hafniaalvei bacteria increased pHSL expression (FIG. 16) as compared tocontrol and gavage with E. coli K12 bacteria. Therefore, E. coli Niessle1917 and Hafnia alvei bacteria activate the mechanism of lipolysis.

Example 7

This example demonstrates the effect of ClpB-expressing Hafnia alvei onhigh fat diet-induced obese mice.

One-month-old male C57B16 mice (Janvier Laboratories) were induced withhigh fat/high carbs diet for 2 weeks. Mice were then intragastricallygavaged with (i) 10⁸ Hafnia alvei AF036 bacteria (expressing ClpB); (ii)10⁸ E. coli K12 bacteria deficient for ClpB; both in Mueller-Hilton (MH)medium or with (iii) MH medium only, as a control. Mice were placedindividually into the BioDAQ cages (Research Diets) and intragastricallygavaged daily for 21 days as indicated.

Induction of obesity by high fat diet was validated by measurement ofmean body weight (FIG. 17A), fat mass (FIG. 17B) and lean mass (FIG.17C) in a group induced and a group non-induced for obesity.

The inventors showed that gavage with any strain of Hafnia alveiClpB-expressing bacteria induced a reduction in weight gain (FIG. 18)and a reduction in fat content (FIG. 19).

Example 8

This example demonstrates the effect of ClpB-expressing Hafnia alvei onobese fa/fa Zucker rats.

Genetically obese fa/fa Zucker rats (Janvier Laboratories) wereacclimated to the animal facility for 1 week and maintained as describedabove. Rats were intragastrically gavaged with (i) 10⁸ Hafnia alveiAF036 bacteria (expressing ClpB); (ii) 10⁸ E. coli K12 bacteriadeficient for ClpB; (iii) 10⁸ E. coli Niessle 1917 bacteria (expressingClpB); all in Mueller-Hilton (MH) medium or with (iv) MH medium only, asa control. Rats were placed individually into the BioDAQ cages (ResearchDiets) and intragastrically gavaged daily for 21 days as indicated.

The results show that body weight gain is mostly decreased in H. alveigroup when compared to rats who received E. coli K12 Δ-C1pB or E. coliNiessle 1917 bacteria, or to control (FIG. 20). Moreover, the lowestcumulative food intake is observed in H. Alvei group in comparison tothe three other groups (FIG. 21).

The inventors also show that fat mass gain is only decreased in H. alveigroup (FIG. 22).

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The invention claimed is:
 1. A method of stimulating weight loss in awarm-blooded subject in need thereof, the method comprisingadministering to the subject an effective amount of Hafnia alvei.
 2. Themethod according to claim 1, wherein administering an effective amountof Hafnia alvei prolongs satiety in the subject in need thereof.
 3. Themethod according to claim 1, wherein administering an effective amountof Hafnia alvei reduces the meal size in the subject in need thereof. 4.The method according to claim 1, wherein administering an effectiveamount of Hafnia alvei reduces food intake in the subject in needthereof.
 5. The method according to claim 1, wherein the subject is notobese.
 6. The method according to claim 1, wherein the subject is obese.7. The method according to claim 1, wherein Hafnia alvei is administeredto the subject in the form of a pharmaceutical composition.
 8. Themethod according to claim 1, wherein Hafnia alvei is administered to thesubject in the form of a food composition.
 9. The method according toclaim 8, wherein the food composition is selected from the groupcomprising a complete food composition, a food supplement, and anutraceutical composition.
 10. The method according to claim 1, whereinHafnia alvei is a probiotic.
 11. The method according to claim 1,wherein Hafnia alvei is inactivated.
 12. The method according to claim8, wherein the food composition comprises at least one prebiotic. 13.The method according to claim 12, wherein at least one prebioticcomprises a dietary fiber.
 14. The method according to claim 1, whereinadministering the effective amount of Hafnia alvei reduces fat mass onlean mass ratio in the subject in need thereof.
 15. The method accordingto claim 1, wherein Hafnia alvei is encapsulated into anenterically-coated, time-released capsule or tablet.
 16. The methodaccording to claim 1, wherein the administration of Hafnia alvei isrepeated 2 to 3 times a day, for one day or more.
 17. The methodaccording to claim 1, wherein Hafnia alvei is administeredsimultaneously or sequentially with one meal of the subject.
 18. Themethod according to claim 1, wherein Hafnia alvei is administered priorto the subject's meal.
 19. The method according to claim 1, wherein thesubject is overweight and has a body mass index (BMI) of between 25 and30.
 20. The method according to claim 1, wherein the subject is obeseand has a body mass index (BMI) that is greater than 30.